Compositions, systems and methods for cell therapy

ABSTRACT

Disclosed herein are compositions and methods for cell therapy comprising an engineered cell. The present invention is directed to a composition for treating a subject having or suspected of having a disease, the composition comprising a modified cell comprising a modified endogenous gene, wherein an endogenous gene or fragment thereof is replaced with a transgene using a CRISPR/Cas9 system to generate the modified endogenous gene, the modified cell having an altered response to a cell signal or stimulus.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/528,296, filed May 19, 2017, which is a national stage filing under35 U.S.C. 371 of International Patent Application No. PCT/US2015/062024,which claims priority to U.S. Provisional Application No. 62/082,315,filed Nov. 20, 2014, which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application includes a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 23, 2015, isnamed 028193-9207-WO00 Sequence Listing.txt and is 8,906 bytes in size.

TECHNICAL FIELD

The present disclosure is directed to compositions and methods for celltherapy comprising an engineered cell.

BACKGROUND

Immunotherapy and regenerative medicine provides the exciting potentialfor cell-based therapies to treat many diseases and restore damagedtissues, but the inability to precisely control cell function haslimited the ultimate success of this field. For over 40 years, genetherapy has been proposed as an approach to cure genetic diseases byadding functional copies of genes to the cells of patients with definedgenetic mutations. However, this field has been limited by the availabletechnologies for adding extra genetic material to human genomes. Inrecent years, the advent of synthetic biology has led to the developmentof technologies for precisely controlling gene networks that determinecell behavior. Several new technologies have emerged for manipulatinggenes in their native genomic context by engineering synthetictranscription factors that can be targeted to any DNA sequence. Thisincludes new technologies that have enabled targeted human geneactivation and repression, including the engineering of transcriptionfactors based on zinc finger proteins, TALEs, and the CRISPR/Cas9system. There remains a need for the ability to precisely regulate anygene as it occurs naturally in the genome, such as the rewiring ofgenetic circuits, as a means to address a variety of diseases anddisorders while circumventing some of the traditional challenges of genetherapy.

SUMMARY

The present invention is directed to a composition for treating asubject having or suspected of having a disease, the compositioncomprising a modified cell comprising a modified endogenous gene,wherein an endogenous gene or fragment thereof is replaced with atransgene using a CRISPR/Cas9 system to generate the modified endogenousgene, the modified cell having an altered response to a cell signal orstimulus.

The present invention is directed to a composition for treating asubject having or suspected of having a disease or disorder, thecomposition comprising a modified cell comprising a modified endogenousgene, wherein an endogenous gene or fragment thereof comprises a signalpeptide and the signal peptide is deleted or knocked out using aCRISPR/Cas9 system to generate the modified endogenous gene, themodified cell having an altered response to a cell signal or stimulus.

The present invention is directed to a method of preventing, treating orameliorating a disease in a subject, the method comprising administeringto the subject a therapeutically effective amount of the disclosedcomposition.

The present invention is directed to a method of generating a modifiedcell comprising a modified endogenous gene, the modified cell having analtered response to a cell signal or stimulus, the method comprisingreplacing an endogenous gene or fragment thereof with a transgene usinga CRISPR/Cas9 system to generate the modified endogenous gene.

The present invention is directed to a method of generating a modifiedcell comprising a modified endogenous gene comprising a signal peptide,the modified cell having an altered response to a cell signal orstimulus, the method comprising deleting or knocking out the signalpeptide using a CRISPR/Cas9 system to generate the modified endogenousgene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of designer cell objectives.

FIG. 2 shows an approach for the development of a “smart” drug deliverysystem for the treatment of arthritis. Induced pluripotent stem cells(iPSCs) are genetically modified to respond to the pro-inflammatorycytokine TNF-a by producing the soluble TNF receptor (sTNFR1), aninhibitor of TNF-a, instead of the degradative enzyme aggrecanase(ADAMTS-5). This tissue-engineered construct can be implantedsubcutaneously, providing an autoregulated factory for anti-inflammatorydrug delivery.

FIG. 3 shows an experimental overview.

FIG. 4 shows an experimental overview.

FIG. 5A shows genomic PCR from clones isolated after single-celldeposition. PCR amplicons represent the presence or absence of exon 2 inthe Illrl locus.

FIG. 5B shows Sanger sequencing from an allele with theCRISPR/Cas9-mediated deletion of exon2 from Illrl. FIG. 5C shows theflow cytometry histograms demonstrating differential levels of Illrlsurface expression in populations derived from each of the Illrl+/+,Illrl+/− and Illrl−/− genotypes. FIG. 5D shows luminescence datacharacterizing the transcriptional activity of NF-icB in Illrl+/+,Illrl+/−, and Illrl−/− cells after a 24 hour treatment with 1 ng/mlIL-la. Bars represent group means±SEM (n=4). Groups not sharing the sameletter are statistically different (p<0.05).

FIGS. 6A-6I show relative gene expression data for Ccl2 (FIG. 6A), 116(FIG. 6B), Elf3 (FIG. 6C), Adamts4 (FIG. 6D), Adamts5 (FIG. 6E), Mmp9(FIG. 6F), Acan (FIG. 6H), and Col2a1 (FIG. 6I) as measured by qRT-PCRto examine the effects of IL-la treatment on engineered cartilagederived from either Il1r1+/+, or Il1r1+/−, or Il1r1−/− cells. Foldchanges were determined relative to a reference group cultured withoutIl-la and by using 18s rRNA as a reference gene. Bars represent groupmeans of fold change±SEM (n=4). Groups not sharing the same letter arestatistically different (p<0.05).

FIGS. 7A-7F show the biochemical analyses of engineered cartilagecomposition. FIG. 7A shows the double-stranded DNA (dsDNA) content. FIG.7B shows the sulfated glycosaminoglycan (sGAG) per DNA. FIG. 7C showsthe total collagen per DNA. FIG. 7D shows the total sGAG per aggregate.FIG. 7E shows the total collagen per aggregate. Bars represent groupmeans±SEM (n=6). *p<0.05 between Illrl+/− and other genotypes. Groupsnot sharing the same letter are statistically different (p<0.05). FIG.7F shows the representative images from safranin-O/fastgreen/hematoxylin staining of 10 μm sections of engineered cartilagetreated with or without 1 ng/ml of IL-1 for 72 hours. Scale bar=500 vtm.

FIGS. 8A-8D show the analysis of media samples collected from engineeredcartilage aggregates cultured with or without 1 ng/ml 1L-1 a for 72hours. FIG. 8A shows the specific MMP activity (n=7). RFU indicatesrelative fluorescence units. FIG. 8B shows the concentration of sGAGmeasured in culture media (n=6). FIG. 8C shows the PGE2 concentration(n=4). FIG. 8D shows the Total nitric oxide concentrations (n=4). Barsrepresent group means±SEM. Groups not sharing the same letter arestatistically different (p<0.05).

FIGS. 9A-9D show a depiction of the re-purposed inflammatory signalingpathway in CRISPR/Cas9-engineered cells. FIG. 9A shows in wild-type (WT)cells, TNF-a signaling through its type 1 receptor initiates a cascadeleading to nuclear translocation and increased transcriptional activityof NE-1B, activating an inflammatory transcriptional program. One generapidly and highly upregulated by cytokine-induced NF-KB activity isCc12 (shown in orange). FIG. 9B shows that a CRISPR/Cas9 RNA-guidednuclease (not depicted) generates a double strand break in theendogenous chromosomal locus near the start codon for Cc12. Provision ofa targeting vector with a transgene flanked by regions homologous to theCc12 locus promotes the use of this template for repair of the damagedallele in a subset of cells. FIG. 9C shows that such alleles would thenbe activated by TNF-a, which would now drive expression of the solubleTNF type 1 receptor (sTNFR1). FIG. 9D shows that upon antagonism ofTNF-a in the microenvironment, signal transduction through the membranereceptor would halt, NF-KB would remain sequestered in the cytoplasm,and expression of the sTNFR1 transgene would autonomously decay uponresolution of the local inflammation.

FIG. 10 shows ethidium bromide-stained agarose gel demonstrating theresult of junction PCR probing for targeted integration of transgenes tothe Cc12 locus. Numbers after abbreviations indicate clone number (e.g.,sTNFR23=clone 23 in which the sTNFR1 transgene was targeted to Cc12). Ineach reaction, wild-type (WT), Cc12-Illra, Cc12-Luc, or Cc12-sTNFR1genomic DNA was used as a template. A 2 Log Ladder (NEB) was run alongwith samples and is shown in the right-most lane.

FIG. 11A shows Cc12 gene expression profile from Wild-type cells.qRT-PCR data showing the expression profile of Cc12 after treatment ofWT cells with various concentrations of TNF-a (n=3). FIG. 11B showscytokine-induced expression of luciferase from endogenous Cc12 Locus (20ng/mL TNF). Two cell lines were engineered to express luciferase fromthe endogenous Cc12 locus and were then stimulated with 20 ng/ml ofTNF-a. Cells were lysed at the indicated time after TNF treatment andluminescence was measured as a readout for Cc12-driven transgeneexpression (n=6).

FIGS. 12A-12D show the profile of 116 expression in response to variousdoses (X-axes) of TNF and across the indicated time points of 4 hrs(FIG. 12A), 12 hrs (FIG. 12B), 24 hrs (FIG. 12C), and 72 hrs (FIG. 12D).Values plotted represent the mean fold change in expression±SEM (n=3) ascompared to matched cells of the same genotype treated with 0 ng/ml TNFand as normalized by the r18S reference gene.

FIG. 13A shows fold change in NF-KB transcriptional activity as measuredby the luminescence signal from NF-KB-dependent firefly luciferaseexpression. Bars represent the mean fold change in relative luminescenceunits (RLU)±SEM of cells treated with 20 ng/ml TNF-a for the indicatedtime as compared to controls cultured with 0 ng/ml TNF-a (n=4-6). FIG.13B shows changes in Cc12-driven expression of the sTNFR1 transgene overtime as measured by qRT-PCR. Values plotted represent the mean foldchange in expression±SEM (n=3) as compared to matched cells of the samegenotype treated with 0 ng/ml TNF-a and as normalized by the r18Sreference gene. The 0 hr time point (shaded) was not measured and isshown for illustration purposes only, as all samples at 0 hrs measure 1by definition. FIG. 13C shows ELISA data showing the concentration ofsTNFR1 protein in culture media in samples treated with the indicatedconcentrations of TNF-a. Samples were collected at the indicated time.Values represent mean±SEM (n=3).

FIGS. 14A-14C show ELISA measurement of transgene product measured inculture media sampled at various 24-hour intervals relative to treatmentwith no cytokine, 0.1 ng/ml IL-1, or 20 ng/ml TNF. FIG. 14A shows DailysTNFR1 secretion before and after cytokine stimulation. sTNFR1 levelsmeasured in culture media conditioned for 24 hrs prior to (DO) and after(D1) cytokine treatment. On DI, cytokine was withdrawn from all samples,and media were collected at 24 hr intervals for the subsequent threedays. FIG. 14B shows sTNFR1 secretion after iterative cytokinestimulation. Cc12-sTNFR1 engineered cells were treated with cytokine,and 24 hrs later, media were collected. Cytokine was then withdrawn for3 days prior to a second and then third stimulation to probe thekinetics of 24-hr sTNFR1 secretion after iterative stimulations. FIG.14C shows IL Ira secretion after iterative cytokine stimulation. Thesame experiment as described in FIG. 14B was performed using Cc12-111raengineered cells, and ELISA was performed on samples to determineprotein levels of Illra secreted into the culture media. Data labelsindicate group average values. Bars represent the mean±SEM (n=3).

FIGS. 15A-15H show relative gene expression data for Cc12 (FIG. 15A),116 (FIG. 15B), Adamts4 (FIG. 15C), Adamts5 (FIG. 15D), Mmp9 (FIG. 15E),Mmp13 (FIG. 15F), Acan (FIG. 15G), and Col2a1 (FIG. 15H) as measured byqRT-PCR to examine the effects of 1 ng/ml IL-1 treatment on engineeredcartilage derived from either WT, Cc12-Luc, or Cc12-Illra cells. Foldchanges were determined relative to a reference group cultured withoutIL-la and by using 18s rRNA as a reference gene. Bars represent groupmeans of fold change±SEM (n=3). Groups not sharing the same letter arestatistically different (p<0.05). Notation of n.s. implies nosignificance for the evaluated gene.

FIGS. 16A-16H show relative gene expression data for Cc12 (FIG. 16A),116 (FIG. 16B), Adamts4 (FIG. 16C), Adamts5 (FIG. 16D), Mmp9 (FIG. 16E),Mmpl 3 (FIG. 16F), Acan (FIG. 16G), and Col2a1 (FIG. 16H) as measured byciRT-PCR to examine the effects of 20 ng/ml TNF treatment on engineeredcartilage derived from either WT, Cc12-Luc, or Cc12-sTNFR1 cells. Foldchanges were determined relative to a reference group cultured withoutIL-la and by using 18s rRNA as a reference gene. Bars represent groupmeans of fold change+SEM (n=3). Groups not sharing the same letter arestatistically different (p<0.05).

FIG. 17A shows sulfated glycosaminoglycan (sGAG) per double-stranded DNAas measured via the dimethylmethylene blue assay in cartilage aggregatesengineered from WT, Cc12-Luc, or Cc12-sTNFR1 cells and maintained incontrol medium or medium supplemented with 20 ng/ml TNF-a for 3 daysafter maturation. FIG. 17B shows sGAG/DNA in cartilage aggregatesengineered from WT, Cc12-Luc, or Cc12-Illra cells and maintained incontrol medium or medium supplemented with 1 ng/ml IL-la for 3 daysafter maturation. FIG. 17C shows sGAG/DNA in cartilage aggregatesengineered from either Cc12-Luc or Ccl2-Illra cells and maintained incontrol medium or medium supplemented with 0.1 ng/ml IL-la for 3 daysafter maturation. FIG. 17D shows Percent change in sGAG content upontreatment with 0.1 ng/ml IL-la relative to 0 ng/ml IL-la controlsamples. Bars represent the mean+SEM (n=3-6). Groups not sharing thesame letter are statistically different (p<0.05).

FIG. 18 shows photomicrographs from Safranin-O/FastGreen/Hematoxylin-stained tissue sections from engineered cartilagesamples. Scale bar=50 um.

DETAILED DESCRIPTION

The present disclosure provides compositions, systems and methods forcell therapy comprising an engineered or modified cell. In particular,target cells are engineered with gene regulatory factors to enhance thetherapeutic effect of various therapies, such as stem cell therapies,for tissue regeneration and treatment of a variety of acute and chronicdiseases and cancer. The modified cell may be implanted into tissue andprovide self-regulated, feedbackcontrol of a therapy, such asanti-cytokine therapy, to the body of a subject. The modified cell maybe engineered to delete pathological cell outputs, possess a syntheticinput-output gene regulatory system, and/or rewire cells to producetherapeutic molecules in response to pathological signals.

The present disclosure provides an innovative method to rewire cellulargene circuits and created a synthetic transcriptional system in a mannerthat allows for the creation of unique and customized cells that cansense and respond to their environment in a pre-programmed way, such asdynamically responding to physiological signals. This distinctapplication of rewiring gene networks in mammalian genomes and cells maycontrol any input-output relationships in cells. The cell's ownmachinery is reprogrammed to detect subtle, dynamic cues within the bodyto actively regulate cell response. Using target cells, such as inducedpluripotent stem cells, intrinsic signaling pathways may be rewired tovirtually “hijack” pathologic signaling cascades (e.g., inflammation) tostimulate controlled therapeutic responses (e.g., anti-inflammatorymolecules), thus creating possibilities for safer and more effectivetreatments for a wide variety of diseases. This self-regulated systemcould be used in controlling tissue growth or regeneration, mediatingtissue repair, or acting as an in situ factory for therapeutic proteindelivery. The engineered, self-regulating cells may be used to controlthe magnitude and duration of biologic therapy for chronic diseases,guide the regeneration of damaged tissues, or produce easily measurablein vivo biomarkers, in which the synthetic transcriptional system can beused to drive expression of reporter molecules.

The compositions and methods that comprise the modified cells, asdescribed herein, provides: (1) Broad applicability—any input and outputcan be targeted for rewiring; similarly, any therapeutic transgene canbe delivered via the synthetic transcriptional system; (2)Specificity—by using gene-editing nucleases, precise modification of thecell's DNA can be performed at a particular site; (3) Flexibility—usingcells, such as iPSCs, allow studies to address diseases associated witha variety of other types of tissues or organs.

1. Definitions

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

“Adeno-associated virus” or “AAV” as used interchangeably herein refersto a small virus belonging to the genus Dependovirus of the Parvoviridaefamily that infects humans and some other primate species. AAV is notcurrently known to cause disease and consequently the virus causes avery mild immune response.

“Binding region” as used herein refers to the region within a nucleasetarget region that is recognized and bound by the nuclease.

“Cancer” as used herein refers to the uncontrolled and unregulatedgrowth of abnormal cells in the body. Cancerous cells are also calledmalignant cells. Cancer may invade nearby parts of the body and may alsospread to more distant parts of the body through the lymphatic system orbloodstream. Cancers include Adrenocortical Carcinoma, Anal Cancer,Bladder Cancer, Brain Tumor, Breast Cancer, Carcinoid Tumor,Gastrointestinal, Carcinoma of Unknown Primary, Cervical Cancer, ColonCancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile DuctCancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor,Intraocular Melanoma Eye Cancer, Gallbladder Cancer, Gastric Cancer(Stomach), Extragonadal Germ Cell Tumor, Gestational TrophoblasticTumor, Head and Neck Cancer, Hypopharyngcal Cancer, Islet CellCarcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, AcuteLymphoblastic Leukemia, Leukemia, Acute Myeloid, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Hairy Cell Leukemia, Lip andOral Cavity Cancer, Liver Cancer, Non-Small Cell Lung Cancer, Small CellLung Cancer, AIDS-Related Lymphoma, Central Nervous System (Primary)Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin's Disease Lymphoma,Non-Hodgkin's Disease Lymphoma, Malignant Mesothelioma, Melanoma, MerkelCell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary,Multiple Myeloma and Other Plasma Cell Neoplasms, Mycosis Fungoides,Myelodysplastic Syndrome, Myeloproliferative Disorders, NasopharyngealCancer, euroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Pancreatic Cancer,Exocrine, Pancreatic Cancer, Islet Cell Carcinoma, Paranasal Sinus andNasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer, Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (cancer of the kidney), Transitional CellRenal Pelvis and Ureter, Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Testicular Cancer,Malignant Thymoma, Thyroid Cancer, Urethral Cancer, Uterine Cancer,Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, and Wilms'Tumor.

“Cell therapy” as used herein refers to a therapy in which cellularmaterial is injected into a patient. The cellular material may beintact, living cells. For example, T cells capable of fighting cancercells via cell-mediated immunity may be injected in the course ofimmunotherapy. Cell therapy is also called cellular therapy orcytotherapy.

“Chronic disease” as used refers to a long-lasting condition that can becontrolled but not cured.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered. The coding sequence may be codonoptimize.

“Complement” or “complementary” as used herein means a nucleic acid canmean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.“Complementarity” refers to a property shared between two nucleic acidsequences, such that when they are aligned antiparallel to each other,the nucleotide bases at each position will be complementary.

“Donor DNA”, “donor template” and “repair template” as usedinterchangeably herein refers to a double-stranded DNA fragment ormolecule that includes at least a portion of the gene of interest. Thedonor DNA may encode a full-functional protein or a partially-functionalprotein.

“Endogenous gene” as used herein refers to a gene that originates fromwithin an organism, tissue, or cell. An endogenous gene is native to acell, which is in its normal genomic and chromatin context, and which isnot heterologous to the cell. Such cellular genes include, e.g., animalgenes, plant genes, bacterial genes, protozoal genes, fungal genes,mitochondrial genes, and chloroplastic genes.

“Functional” and “full-functional” as used herein describes protein thathas biological activity. A “functional gene” refers to a genetranscribed to mRNA, which is translated to a functional protein.

“Fusion protein” as used herein refers to a chimeric protein createdthrough the joining of two or more genes that originally coded forseparate proteins. The translation of the fusion gene results in asingle polypeptide with functional properties derived from each of theoriginal proteins.

“Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence that encodes a protein. The codingsequence includes initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of the individual to whomthe nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operably linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

“Genome editing” as used herein refers to changing a gene. Genomeediting may include correcting or restoring a mutant gene. Genomeediting may include knocking out a gene, such as a mutant gene or anormal gene. Genome editing may be used to treat disease or enhancetissue repair by changing the gene of interest.

The term “heterologous” as used herein refers to nucleic acid comprisingtwo or more subsequences that are not found in the same relationship toeach other in nature. For instance, a nucleic acid that is recombinantlyproduced typically has two or more sequences from unrelated genessynthetically arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source. Thetwo nucleic acids are thus heterologous to each other in this context.When added to a cell, the recombinant nucleic acids would also beheterologous to the endogenous genes of the cell. Thus, in a chromosome,a heterologous nucleic acid would include a non-native (non-naturallyoccurring) nucleic acid that has integrated into the chromosome, or anon-native (non-naturally occurring) extrachromosomal nucleic acid.Similarly, a heterologous protein indicates that the protein comprisestwo or more subsequences that are not found in the same relationship toeach other in nature (e.g., a “fusion protein,” where the twosubsequences are encoded by a single nucleic acid sequence).

“Homology-directed repair” or “HDR” as used interchangeably hereinrefers to a mechanism in cells to repair double strand DNA lesions whena homologous piece of DNA is present in the nucleus, mostly in G2 and Sphase of the cell cycle. HDR uses a donor DNA template to guide repairand may be used to create specific sequence changes to the genome,including the targeted addition of whole genes. If a donor template isprovided along with the site specific nuclease, such as with aCRISPR/Cas9-based systems, then the cellular machinery may repair thebreak by homologous recombination, which is enhanced several orders ofmagnitude in the presence of DNA cleavage. When the homologous DNA pieceis absent, non-homologous end joining may take place instead.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Immunotherapy” as used herein refers to the treatment of disease byinducing, enhancing, or suppressing an immune response. Immunotherapiesdesigned to elicit or amplify an immune response are classified asactivation immunotherapies, while immunotherapies that reduce orsuppress are classified as suppression immunotherapies.

“Non-homologous end joining (NHEJ) pathway” as used herein refers to apathway that repairs double-strand breaks in DNA by directly ligatingthe break ends without the need for a homologous template. Thetemplate-independent re-ligation of DNA ends by NHEJ is a stochastic,error-prone repair process that introduces random micro-insertions andmicro-deletions (indels) at the DNA breakpoint. This method may be usedto intentionally disrupt, delete, or alter the reading frame of targetedgene sequences. NHEJ typically uses short homologous DNA sequencescalled microhomologies to guide repair. These microhomologies are oftenpresent in single-stranded overhangs on the end of double-strand breaks.When the overhangs are perfectly compatible, NHEJ usually repairs thebreak accurately, yet imprecise repair leading to loss of nucleotidesmay also occur, but is much more common when the overhangs are notcompatible.

“Nuclease mediated NHEJ” as used herein refers to NHEJ that is initiatedafter a nuclease, such as a Cas9, cuts double stranded DNA.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid may be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that may hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of dcoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

Nucleic acid or amino acid sequences are “operably linked” (or“operatively linked”) when placed into a functional relationship withone another. For instance, a promoter or enhancer is operably linked toa coding sequence if it regulates, or contributes to the modulation of,the transcription of the coding sequence. Operably linked DNA sequencesare typically contiguous, and operably linked amino acid sequences aretypically contiguous and in the same reading frame. However, sinceenhancers generally function when separated from the promoter by up toseveral kilobases or more and intronic sequences may be of variablelengths, some polynucleotide elements may be operably linked but notcontiguous. Similarly, certain amino acid sequences that arenon-contiguous in a primary polypeptide sequence may nonetheless beoperably linked due to, for example folding of a polypeptide chain. Withrespect to fusion polypeptides, the terms “operatively linked” and“operably linked” can refer to the fact that each of the componentsperforms the same function in linkage to the other component as it wouldif it were not so linked.

“Induced pluripotent stem cells” or “iPSCs” as used interchangeablyherein refers to a type of pluripotent stem cell that can beartificially derived from a non-pluripotent cell, typically an adultsomatic cell, by inducing a “forced” expression of certain genes andtranscription factors.

A “progenitor cell” as used herein refers to a biological cell that,like a stem cell, has a tendency to differentiate into a specific typeof cell, but is already more specific than a stem cell. While stem cellscan replicate indefinitely, progenitor cells can divide only a limitednumber of times.

“Promoter” as used herein means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which may be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (naturally occurring) form of the cell orexpress a second copy of a native gene that is otherwise normally orabnormally expressed, under expressed or not expressed at all.

“Site-specific nuclease” as used herein refers to an enzyme capable ofspecifically recognizing and cleaving DNA sequences. The site-specificnuclease may be engineered. Examples of engineered site-specificnucleases include zinc finger nucleases (ZFNs), TAL effector nucleases(TALENs), and CR1SPR/Cas9-based systems.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. The subject orpatient may be undergoing other forms of treatment.

“Target gene” as used herein refers to any nucleotide sequence encodinga known or putative gene product. The target gene may be an endogenousgene involved in a disease and/or cellular pathway.

“Target region” as used herein refers to the region of the target geneto which the site-specific nuclease is designed to bind and cleave.

“Transgene” as used herein refers to a gene or genetic materialcontaining a gene sequence that has been isolated from one organism andis introduced into a different organism. This non-native segment of DNAmay retain the ability to produce RNA or protein in the transgenicorganism, or it may alter the normal function of the transgenicorganism's genetic code. The introduction of a transgene has thepotential to change the phenotype of an organism.

“Treat”, “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease, or one or more symptoms of such disease, to which such termapplies. Depending on the condition of the subject, the term also refersto preventing a disease, and includes preventing the onset of a disease,or preventing the symptoms associated with a disease. A treatment may beeither performed in an acute or chronic way. The term also refers toreducing the severity of a disease or symptoms associated with suchdisease prior to affliction with the disease. Such prevention orreduction of the severity of a disease prior to affliction refers toadministration of an antibody or pharmaceutical composition of thepresent invention to a subject that is not at the time of administrationafflicted with the disease. “Preventing” also refers to preventing therecurrence of a disease or of one or more symptoms associated with suchdisease. “Treatment” and “therapeutically,” refer to the act oftreating, as “treating” is defined above.

“Stem cells” as used herein refers to an undifferentiated cell of amulticellular organism that is capable of giving rise to indefinitelymore cells of the same type, and from which certain other kinds of cellarise by differentiation. Stem cells can differentiate into specializedcells and can divide (through mitosis) to produce more stem cells. Theyare found in multicellular organisms. In mammals, there are two broadtypes of stem cells: embryonic stem cells, which are isolated from theinner cell mass of blastocysts, and adult stem cells, which are found invarious tissues. In adult organisms, stem cells and progenitor cells actas a repair system for the body, replenishing adult tissues. In adeveloping embryo, stem cells can differentiate into all the specializedcells, such as ectoderm, endoderm and mesoderm, but also maintain thenormal turnover of regenerative organs, such as blood, skin, orintestinal tissues.

“Suicide gene” as used herein refers to a gene that will cause a cell tokill itself through apoptosis. Activation of these genes may be due tomany processes, but the main cellular “switch” to induce apoptosis isthe p53 protein. Stimulation or introduction (through gene therapy) ofsuicide genes may be used to treat cancer or other proliferativediseases by making cancer cells more vulnerable, more sensitive tochemotherapy. Parts of genes expressed in cancer cells are attached toother genes for enzymes not found in mammals that can convert a harmlesssubstance into one that is toxic to the tumor. The suicide genes thatmediate this sensitivity may encode for viral or bacterial enzymes thatconvert an inactive drug into toxic antimetabolites that inhibit thesynthesis of nucleic acid.

“T cell” or “T lymphocyte” as used interchangeably herein refers to acell derived from thymus among lymphocytes involved in an immuneresponse.

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes may be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes may besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids may also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector may be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid.

2. Compositions for Cell Therapy

Provided herein are compositions for use in cell therapy. Thecompositions include a modified cell having a modified endogenous gene.The modified cells may be engineered or modified ex vivo with engineeredtranscriptional regulators or other genome engineering tools, such assite specific nucleases, targeted to one or more specific endogenousgenes of interest or target genes. The modified cells may have syntheticactivators or repressors of genes that control stem cell survival,proliferation, engraftment, migration, homing, and/or response toendogenous inflammatory, immunomodulatory, or morphogenetic signals. Forexample, stem cells may be reprogrammed through genome engineering forautonomously regulated anti-cytokine therapy. In some embodiments, themodified cells may have a reprogrammed checkpoint inhibitor signalingpathway.

The site-specific nuclease may bind and cleave a target endogenous geneor locus in the genome of a cell. For example, the target endogenousgene or locus may be Ccl2, ADAMTS-5 or IL Irl gene. The modification maybe by delivery of nucleic acids encoding the gene-modifying agent,including viral transduction or transfection of plasmid DNA or mRNA, orby direct treatment with the zinc finger protein, TALE protein, orCRISPR/Cas9 protein:RNA complex. In some embodiments, theCRISPR/Cas9-based system, as described below, may be used to introducesite-specific double strand breaks at targeted genomic loci to modifythe endogenous gene. In some embodiments, the coding region of theendogenous gene is replaced with the coding region of a transgene usingthe CRISPR/Cas9-based system to generate the modified endogenous gene.In some embodiments, the endogenous gene or fragment thereof is deletedor knocked out using the CRISPR/Cas9-based system to generate themodified endogenous gene.

The modified cell may be engineered to delete pathological cell outputs.Functional deficiencies or complete knock-out are generated of theproteins encoded by the targeted endogenous gene in cells, such as stemcells. In some embodiments, the chromosomal loci of genes involved inthe inflammatory response of cells are targeted.

The modified cell may be engineered to possess a synthetic input-outputgene regulatory system. Synthetic, self-regulating transcriptionalsystem is generated that is activated by a specific input, e.g.,inflammatory cytokines, insulin, glucose, etc., or any other cellularfeedback system, natural, and artificial. In some embodiments, thesynthetic, self-regulating transcriptional system is activated byinflammatory cytokines. Starting with iPSCs, an artificial promoterdriving co-expression of luciferase, as well as synthetic transcriptionfactors designed to confer important engineering controls to the systemmay be engineered. The artificial promoter may include tandem repeats ofknown binding sites of endogenous transcription factors involved indriving inflammatory transcriptional programs, such as members of therel/NF-xl3 family. Moreover, binding sites recognized by the synthetictranscription factors allow fine-tuning of the sensitivity, dynamicrange, temporal responsiveness, and memory of the transcriptionalcontrol system. A sensitive luciferase-based assay may be used toiteratively tune each of these critical design parameters for optimalperformance of the cellular response.

The modified cell may be engineered to rewire cells to producetherapeutic molecules in response to pathological signals. In someembodiments, the therapeutic molecule may be an anti-inflammatory,anti-cytokine, pro-anabolic, or analgesic molecule. For example, thetherapeutic molecule may be encoded by a gene whose product affects theinflammatory pathway and/or affects the activity or the pathways ofsignalling of cytokines.

3. Altered Response to Cell Signal or Stimulus

The modified cell has an altered response to cell signal or stimulus.Using induced pluripotent stem cells, intrinsic signaling pathways maybe rewired to virtually “hijack” pathologic signaling cascades, forexample, inflammation, to stimulate controlled therapeutic responses,for example, anti-inflammatory molecules, creating possibilities forsafer and more effective treatments for a wide variety of diseases. Insome embodiments, normal cellular responses may be reprogrammed not onlyto control the differentiation state of these cells, but also to rewirethe genetic circuitry of the cells to define novel input-outputrelationships.

In some embodiments, the altered response to the cell signal or stimulusmay involve the activation of the transgene which activates ordownregulates a signaling pathway in response to the cell signal orstimulus as compared to the response to the cell signal or stimulus bythe unmodified endogenous gene. For example, the activation of thetranscription factor may activate an anti-inflammatory response whilethe unmodified endogenous gene may activate an inflammatory response.The inflammatory cell signaling network may be either disrupted orreprogrammed in order to generate engineered tissues capable ofmodulating an effective response against pro-inflammatory cytokines.

In some embodiments, the altered response to the cell signal or stimulusmay involve the functional deficiency or complete knock-out of theprotein encoded by the target endogenous gene or fragment thereof. Thealtered response proteins may be a decrease in responsiveness of themodified endogenous gene to the cell signal or stimuli compared to anunmodified endogenous gene.

a) Cell Signal or Stimulus

The compositions may be used to design synthetic, self-regulatingtranscriptional system that is activated by a specific input, e.g.,inflammatory cytokines, insulin, glucose, etc., or any other cellularfeedback system, natural, and artificial. For example, the cell signalor stimulus (“the input stimulus”) may be any chemical or mechanicalsignal, such as soluble factors, cell-cell, or cell-matrix interactions;inflammatory stimulus such as stimulation with various concentrationsand durations of TNF. Cell signaling that is redirected may be used torewire the genetic program activated by any stimulus, i.e., physicalsignals, hormones, growth factors, inflammatory or anti-inflammatorycytokines, transcription factors, etc.

b) Cytokine-Inducible

The modified cells may include a feedback control system for cell-baseddelivery of inhibitors of tumor necrosis factor alpha (TNF), apro-inflammatory cytokine that plays a key role in a number ofautoimmune disorders, particularly rheumatoid arthritis. Modified cellsmay be generated that respond directly to harmful inflammatory cues byautomatically producing anti-inflammatory molecules to treat autoimmunediseases, such as rheumatoid arthritis. This disease is characterized bypainful flares that are mediated by inflammatory cytokines andultimately lead to destruction of the joint, including the articularcartilage. Current therapies for this disease involve high andunregulated doses of anti-cytokine therapies, which are associated withsignificant side effects and risks. The development of modified cellsmay regenerate articular cartilage, while intrinsically protectingagainst inflammation-mediated degradation.

A promoter of an endogenous gene may be utilized in the modified cellsto regulate anti-cytokine therapy in an autonomous, real-time fashion.Features making an endogenous locus a suitable candidate for thisendeavor include the following: (1) The co-opted gene must becytokine-inducible. (2) Basal expression from the endogenous gene mustbe low to prevent unwanted expression of anti-cytokine therapy.Furthermore, low basal expression is required to permit detection ofincreased local cytokine levels by engineered cells. (3) Expressionkinetics of the gene must be sufficient to generate a rapid responsesuch that the inflammatory program is reversible by production ofanti-cytokine therapy. (4) Cytokine-induced gene expression must persistfor an adequate period of time to ensure the response is sufficientlyrobust to combat persistent inflammatory cues. (5) The gene should beinduced by a variety of cell types in response to inflammatory cues. (6)Disruption of at least one allele of this gene ought to have no negativeconsequences on overall cellular function.

For example, pluripotent stem cells may be modified with the prescribedfeature of inflammatory cytokine resistance by performing targetedaddition of therapeutic transgenes to the cytokine-responsive Cc12locus. Transgene expression from engineered cells may befeedback-controlled with rapid on/off dynamics and may be adequate tomitigate the inflammatory effects of physiologic concentrations of bothIL-1 a and TNF-a in the context of precursor cells cultured in monolayeras well as in engineered tissues such as cartilage.

Modified cells may provide autonomous and dynamically regulatedproduction of therapeutic molecules in response to a pre-programmedbiological signal. In some embodiments, mammalian stem cells aremodified to provide feedback control for self-regulated delivery of aninhibitor of the pro-inflammatory cytokine, tumor necrosis factor alpha(TNF), as a therapy for rheumatoid arthritis, a progressive autoimmunedisease that leads to painful joint destruction and disability. Thissystem is advantageous for being a self-regulating drug delivery system,as anti-TNF therapies are highly effective in many patients but areadministered through regular injections at very high doses, increasingsusceptibility to infections as well as the risk of cardiovasculardisease, hepatitis, and cancer. In this regard, the compositioncomprising the modified cell may include a drug delivery system thatbiologically senses TNF levels and responds dynamically with appropriatelevels of a TNF inhibitor for the treatment of rheumatoid arthritis andother autoimmune diseases. A tissue implant comprised of the compositionof modified cells custom-designed with a precisely engineered,feedback-controlled gene circuit may control production of ananti-inflammatory therapy to rapidly and automatically protect the joint(and the whole body) from TNF-mediated damage.

4. Endogenous Target Gene

The modified cell includes a modified endogenous gene, wherein anendogenous gene or fragment thereof is replaced with a transgene using aCRISPR/Cas9 system to generate the modified endogenous gene that arefunctional deficiencies or complete knock-out of the proteins coded bythe targeted genes in the cells. Endogenous target genes include, butare not limited to, a variety of growth factor, inflammatory mediators,and transcription factors, including but not limited to genes encodingCc12, VEGF-A, IL-2, IFN, IL-1Ra, IL-1R2, IL-6, IL-17, ILlrl, telomerase,TNF, IFN, p21, TNFR1, CTLA4, PD-1, certain metalloproteinases (MMPs),such as MMP2, MMP9, and MMP13, ADAMTS, such as aggrecanase (ADAMTS5) andother genes in the inflammatory, pain, or catabolic pathways.

The endogenous target gene may be a gene that is activated by acytokine, such as TNF. There are three broad groups based on geneinduction profiles: class I, II, and III genes (Hao and Baltimore, NatImmunol. (2009) 10:281-288). Class 1 genes responded to TNF early, andtranscripts decayed quickly irrespective of resolution of theinflammatory assault. Examples of Class I genes include Atf3, Axudl,Btg2, c-Fos, c-Jun, Cxcl 1, Cxcl2, Ednl, Ereg, Fos, Gadd45b, Ier2, Ter3,Ifrdl, II Ib, 116, Irfl, Junb, Lif, Nflcbia, Nfkbiz, Ptgs2, Slc25a25,Sqstml, Tieg, Tnf, Tnfaip3, and Zfp36. Class II genes responded aslate-early mediators and were characterized by a robust response withlevels of expression that persisted above baseline only if inflammatorystimuli were also sustained. Upon withdrawal of stimulant, expression ofclass II genes declined significantly but remained elevated over basalexpression. Examples of Class II genes include Birc2, Cc12, Cc120, Cc17,Cebpd, Ch25h, CSF1, Cx3c11, Cxcl10, Cxcl5, Gch, Icaml, Ifi47, Ifngr2,MmplO, Nfkbie, Npall, p21, Relb, Ripk2, Rndl, Slpr3, Stx11, Tgtp, Tlr2,Tmem140, Tnfaip2, Tnfrsf6, and Vcaml. Class III genes were moregradually induced by TNF stimulation and generally continued toaccumulate throughout the course of experiments (24 hr), even if TNF wasremoved. Examples of Class III genes include 1110004C05Rik (GenBankaccession number BC010291), Abcal, A1561871 (GenBank accession numberBI143915), AI882074 (GenBank accession number BB730912), Artsl, AW049765(GenBank accession number BCO26642.1), C3, Casp4, Ccl5, Cc19, Cdsn,Enpp2, Gbp2, H2-D1, H2-K, H2-L, Ifitl, Ill3ral, Illrll, Lcn2, Lhfp12,LOC677168 (GenBank accession number AK019325), Mmp13, Mmp3, Mt2, Nafl,Ppicap, Prnd, PsmbIO, Saa3, Serpina3g, Serpinfl, Sod3, Statl, Tapbp,U90926 (GenBank accession number NM 020562), and Ubd. The modulation ofthese genes may enhance the angiogenic, immunomodulatory, andproliferative potential of the implanted modified cells.

Biological systems often function reliably in diverse environmentsdespite internal or external perturbations. This behavior is oftencharacterized as “robustness.” Much of this robustness can be attributedto the control of gene expression through complex cellular networks.These networks are known to consist of various regulatory modules,including feedback and feed-forward regulation and cell-cellcommunication. With these basic regulatory modules and motifs,researchers are now constructing artificial networks that mimic natureto gain fundamental biological insight and understanding. In addition,other artificial networks that are engineered with novel functions willserve as building blocks for future practical applications. Theseefforts form the foundation of the recent emergence of syntheticbiology. These artificial networks are interchangeably called “syntheticgene circuits” or “engineered gene circuits.” The modified cells mayhave engineered switches, oscillators logic gates, metabolic control,reengineered translational machinery, population control and patternformation using natural or synthetic cell-cell communication,reengineered viral genome, and hierarchically complex circuits builtupon smaller, well-characterized functional modules. In someembodiments, the target endogenous gene may be any gene that is involvedin a complex cellular network.

The endogenous target gene may be gene involved in checkpoint signalingpathway, such as an inhibitory checkpoint molecule. Examples include,but are not limited to, A2AR (Adenosine A2A receptor), B7-H3 (alsocalled CD276), B7-H4 (also called VTCN1), BTLA (B and T LymphocyteAttenuator; also called CD272), CTLA-4 (CytotoxicT-Lymphocyte-Associated protein 4; also called CD152), IDO (Indolcamine2,3-dioxygenase) KIR (Killer-cell Immunoglobulin-like Receptor), LAG3(Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1 (PD-1)receptor), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), andVISTA (V-domain Ig suppressor of T cell activation).

By targeting the transgenes to the start codon of the endogenous targetgene, many of the endogenous regulatory features associated withendogenous target gene expression are preserved, including the distaland proximal regulatory regions. As such, the re-purposed endogenoustarget gene promoter may endow engineered cells with the capacity tosubstantially upregulate transgene expression in an inducible manner.This upregulation may be both dose- and time-dependent and transient innature.

(1) Cc12 or ADAMTS-5

The CRISPR/Cas9 system may be used to produce stem cells with programmedresponses to inflammatory signaling. In these iPSCs, pro-inflammatorycytokine receptors may be kept intact; however, targeted gene additionto a highly responsive, inflammation-inducible locus may be performed inorder to re-purpose a constituent of the inflammatory transcriptionalprogram. An endogenous, inflammation-inducible gene may be co-opted toengineer stem cells capable of auto-regulating the production ofbiologic therapies that effectively counteract degeneration incited bypro-inflammatory cytokines. In some embodiments, the IL-1- andTNF-regulated chemokine (C-C motif) ligand 2 (Cc12) gene may be co-optedby replacing the components of the Cc12 protein coding sequence withthat of the cytokine antagonists IL-1 receptor antagonist (Ill ra) orthe type I soluble TNF receptor (sTNFR1) (see FIG. 3). In someembodiments, ADAMTS-5 may be co-opted by replacing the components of theADAMTS-5 protein coding sequence with that of the cytokine antagonistsIL-1 receptor antagonist (Illra) or the type I soluble TNF receptor(sTNFR1).

Engineered cells may express the endogenous promoter-transgeneconstruct, such as a Cc12-driven transgene construct, in acytokine-inducible manner. Cells may autonomously regulate transgeneexpression in accordance with the degree of inflammation they detected:upon withdrawal of cytokine, Cc12-driven transgene expression mayattenuate the cytokine assault and may lead to decay in expression.Cartilage tissue engineered from these reprogrammed cells may be capableof protecting itself from in vitro treatment of IL-1 and TNF-a thatproved sufficient to degrade cartilage generated from control cells.

Cc12 may be a target locus for controlling transgene expression becauseof its temporal pattern associated with its cytokine-inducibleexpression profile. Cc12 was a gene behaving with rapid inductionkinetics similar to group I genes, whereas it displayed an expressionprofile after the immediate early phase of induction more akin to agroup II gene, whose persistence depended on continued exposure toinflammatory cues. By targeting the transgenes to the Cc12 start codon,many of the endogenous regulatory features associated with Cc12expression are preserved, including the distal and proximal regulatoryregions encompassing two NF-KB regulatory elements as well as the SP1and AP-1 binding sites. As such, the re-purposed Cc12 promoter may endowengineered cells with the capacity to substantially upregulate transgeneexpression in an inflammation-inducible manner. This upregulation may beboth dose- and time-dependent and transient in nature.

In some embodiments, the response of the modified cells may be based ontheir differentiation status, lineage commitment or cell number. Byperforming targeted integration to the Cc12 locus, the transcriptionalcircuitry associated with inflammatory signaling is rewired in iPSCs.Tissues derived from engineered cells were capable of combating thedegenerative effects of cytokine treatment. Genome engineeringfacilitated the rewiring of endogenous cell circuits in order to definenovel input/output relationships between inflammatory mediators andtheir antagonists, achieving therapeutic benefit coupled to a rapidlyresponding, auto-regulated system. Gene-edited iPSCs may bedifferentiated toward the chondrocyte lineage and endowed with theability to autonomously regulate the production of anti-cytokine therapyat levels adequate to confer tissue-level protection against physiologicand supra-physiologic concentrations of cytokine.

(2) Illrl

Modified cells may have a prescribed feature of resistance tointerleukin-1 (IL-1) signaling. The CRISPR/Cas9-based system may includeCas9 and at least one gRNA to target the Illrl gene (see FIG. 4). Insome embodiments, the target region may be within or in proximity ofexon 2 and the IL lrl gene may be modified by deleting the signalpeptide. For example, targeted deletion of the IL-1 type I receptor(Illrl) signal peptide sequence may be implemented in inducedpluripotent stem cells (iPSCs). The cartilage derived from stem cellswith targeted disruption of the Illrl gene may resist degradation drivenby IL-1.

5. Transgenes

The modified endogenous gene or fragment thereof may be replaced with atransgene. In some embodiments, the coding region of the endogenous geneis replaced with the coding region of the transgene and the codingregion of the transgene is operably linked to the promoter of theendogenous gene. This type of system may integrate multiple biologicalinputs to various preprogrammed responses by directly targetingtransgenes to inducible, endogenous loci using the efficient and highlyspecific CRISPR/Cas9 genome engineering technology. In this manner, thelimitations associated with predicting regulatory features in a geneticlocus such as distal enhancers are avoided as the coding region of theendogenous gene is replaced. In addition, this approach abrogates theneed to consider limitations on packaging efficiency, as the entireregulatory region need not be packaged in a gene delivery vector.Moreover, by performing targeted integration, this strategy absolvesconcerns associated with random, uncontrolled insertion of proviruswithin the host genome. The transgene or output may be a therapeuticgrowth factor, a transcriptional regulator, an extracellular matrix(ECM) protein, an anti-inflammatory protein, or a biomarker used tomonitor treatment efficacy or disease progression. For example, thetransgene may be sTNFR1, IL-1Ra, IL6, IkB-alph, IL-10, a suicide gene,or a matrix degrading enzyme, such as a matrix metalloproteinase (MMP).

6. CRISPR System

“Clustered Regularly Interspaced Short Palindromic Repeats” and“CRISPRs”, as used interchangeably herein refers to loci containingmultiple short direct repeats that are found in the genomes ofapproximately 40% of sequenced bacteria and 90% of sequenced archaea.The CRISPR system is a microbial nuclease system involved in defenseagainst invading phages and plasmids that provides a form of acquiredimmunity. The CRISPR loci in microbial hosts contain a combination ofCRISPR-associated (Cas) genes as well as non-coding RNA elements capableof programming the specificity of the CRISPR-mediated nucleic acidcleavage. Short segments of foreign DNA, called spacers, areincorporated into the genome between CRISPR repeats, and serve as a‘memory’ of past exposures. Cas9 forms a complex with the 3′ end of thesgRNA, and the protein-RNA pair recognizes its genomic target bycomplementary base pairing between the 5′ end of the sgRNA sequence anda predefined 20 bp DNA sequence, known as the protospacer. This complexis directed to homologous loci of pathogen DNA via regions encodedwithin the crRNA, i.e., the protospacers, and protospacer-adjacentmotifs (PAMs) within the pathogen genome. The non-coding CRISPR array istranscribed and cleaved within direct repeats into short crRNAscontaining individual spacer sequences, which direct Cas nucleases tothe target site (protospacer). By simply exchanging the 20 bprecognition sequence of the expressed sgRNA, the Cas9 nuclease can bedirected to new genomic targets. CRISPR spacers are used to recognizeand silence exogenous genetic elements in a manner analogous to RNAi ineukaryotic organisms.

Three classes of CRISPR systems (Types I, II and III effector systems)are known. The Type II effector system carries out targeted DNAdouble-strand break in four sequential steps, using a single effectorenzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type IIIeffector systems, which require multiple distinct effectors acting as acomplex, the Type II effector system may function in alternativecontexts such as eukaryotic cells. The Type II effector system consistsof a long pre-crRNA, which is transcribed from the spacer-containingCRISPR locus, the Cas9 protein, and a tracrRNA, which is involved inpre-crRNA processing. The tracrRNAs hybridize to the repeat regionsseparating the spacers of the pre-crRNA, thus initiating dsRNA cleavageby endogenous RNase III. This cleavage is followed by a second cleavageevent within each spacer by Cas9, producing mature crRNAs that remainassociated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNAcomplex.

The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches forsequences matching the crRNA to cleave. Target recognition occurs upondetection of complementarity between a “protospacer” sequence in thetarget DNA and the remaining spacer sequence in the crRNA. Cas9 mediatescleavage of target DNA if a correct protospacer-adjacent motif (PAM) isalso present at the 3′ end of the protospacer. For protospacertargeting, the sequence must be immediately followed by theprotospacer-adjacent motif (PAM), a short sequence recognized by theCas9 nuclease that is required for DNA cleavage. Different Type IIsystems have differing PAM requirements. The S. pyogenes CRISPR systemmay have the PAM sequence for this Cas9 (SpCas9) as 5′-NRG-3′, where Ris either A or G, and characterized the specificity of this system inhuman cells. A unique capability of the CRISPR/Cas9 system is thestraightforward ability to simultaneously target multiple distinctgenomic loci by co-expressing a single Cas9 protein with two or moresgRNAs. For example, the Streptococcus pyogenes Type II system naturallyprefers to use an “NGG” sequence, where “N” can be any nucleotide, butalso accepts other PAM sequences, such as “NAG” in engineered systems(Hsu et al., Nature Biotechnology (2013) doi:10.1038/nbt.2647).Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9)normally has a native PAM of NNNNGATT, but has activity across a varietyof PAMs, including a highly degenerate NNNNGNNN PAM (Esvelt et al.Nature Methods (2013) doi:10.1038/nmeth.2681).

7. CRISPR/Cas9-Based System

An engineered form of the Type II effector system of Streptococcuspyogenes was shown to function in human cells for genome engineering. Inthis system, the Cas9 protein was directed to genomic target sites by asynthetically reconstituted “guide RNA” (“gRNA”, also usedinterchangeably herein as a chimeric single guide RNA (“sgRNA”)), whichis a crRNA-tracrRNA fusion that obviates the need for RNase III andcrRNA processing in general. Provided herein are CRISPR/Cas9-basedengineered systems for use in genome editing. The CRISPR/Cas9-basedengineered systems may be designed to target any gene, including genesinvolved in a genetic disease, aging, tissue regeneration, or woundhealing. The CRISPR/Cas9-based systems may include a Cas9 protein orCas9 fusion protein and at least one gRNA. The Cas9 fusion protein may,for example, include a domain that has a different activity that what isendogenous to Cas9, such as a transactivation domain.

a) Cas9

The CRISPR/Cas9-based system may include a Cas9 protein or a Cas9 fusionprotein. Cas9 protein is an endonuclease that cleaves nucleic acid andis encoded by the CRISPR loci and is involved in the Type II CRISPRsystem. The Cas9 protein may be from any bacterial or archaea species,such as Streptococcus pyogenes. The Cas9 protein may be mutated so thatthe nuclease activity is inactivated. An inactivated Cas9 protein fromStreptococcus pyogenes (iCas9, also referred to as “dCas9”) with noendonuclease activity has been recently targeted to genes in bacteria,yeast, and human cells by gRNAs to silence gene expression throughsteric hindrance. As used herein, “iCas9” and “dCas9” both refer to aCas9 protein that has the amino acid substitutions D10A and H840A andhas its nuclease activity inactivated.

b) Cas9 Fusion Protein

The CRISPR/Cas9-based system may include a fusion protein. The fusionprotein may comprise two heterologous polypeptide domains, wherein thefirst polypeptide domain comprises a Cas protein and the secondpolypeptide domain has nuclease activity that is different from thenuclease activity of the Cas9 protein. The fusion protein may include aCas9 protein or a mutated Cas9 protein, as described above, fused to asecond polypeptide domain that has nuclease activity. A nuclease, or aprotein having nuclease activity, is an enzyme capable of cleaving thephosphodiester bonds between the nucleotide subunits of nucleic acids.Nucleases are usually further divided into endonucleases andexonucleases, although some of the enzymes may fall in both categories.Well known nucleases are deoxyribonuclease and ribonuclease.

c) gRNA

The gRNA provides the targeting of the CRISPR/Cas9-based system. ThegRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. ThesgRNA may target any desired DNA sequence by exchanging the sequenceencoding a 20 bp protospacer which confers targeting specificity throughcomplementary base pairing with the desired DNA target. gRNA mimics thenaturally occurring crRNA:tracrRNA duplex involved in the Type IIEffector system. This duplex, which may include, for example, a42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide forthe Cas9 to cleave the target nucleic acid. The “target region”, “targetsequence” or “protospacer” as used interchangeably herein refers to theregion of the target gene to which the CRISPR/Cas9-based system targets.The CRISPR/Cas9-based system may include at least one gRNA, wherein thegRNAs target different DNA sequences. The target DNA sequences may beoverlapping. The target sequence or protospacer is followed by a PAMsequence at the 3′ end of the protospacer. Different Type II systemshave differing PAM requirements. For example, the Streptococcus pyogenesType II system uses an “NGG” sequence, where “N” can be any nucleotide.

The gRNA may target any nucleic acid sequence such as an endogenousgene, as discussed above. The CRISPR/Cas9-based system may use gRNA ofvarying sequences and lengths. The number of gRNA administered to thecell may be at least 1 gRNA, at least 2 different gRNA, at least 3different gRNA at least 4 different gRNA, at least 5 different gRNA, atleast 6 different gRNA, at least 7 different gRNA, at least 8 differentgRNA, at least 9 different gRNA, at least 10 different gRNAs, at least11 different gRNAs, at least 12 different gRNAs, at least 13 differentgRNAs, at least 14 different gRNAs, at least 15 different gRNAs, atleast 16 different gRNAs, at least 17 different gRNAs, at least 18different gRNAs, at least 18 different gRNAs, at least 20 differentgRNAs, at least 25 different gRNAs, at least 30 different gRNAs, atleast 35 different gRNAs, at least 40 different gRNAs, at least 45different gRNAs, or at least 50 different gRNAs. The number of gRNAadministered to the cell may be between at least 1 gRNA to at least 50different gRNAs, at least 1 gRNA to at least 45 different gRNAs, atleast 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, atleast 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, atleast 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, atleast 4 gRNAs to at least 50 different gRNAs, at least 4 different gRNAsto at least 45 different gRNAs, at least 4 different gRNAs to at least40 different gRNAs, at least 4 different gRNAs to at least 35 differentgRNAs, at least 4 different gRNAs to at least 30 different gRNAs, atleast 4 different gRNAs to at least 25 different gRNAs, at least 4different gRNAs to at least 20 different gRNAs, at least 4 differentgRNAs to at least 16 different gRNAs, at least 4 different gRNAs to atleast 12 different gRNAs, at least 4 different gRNAs to at least 8different gRNAs, at least 8 different gRNAs to at least 50 differentgRNAs, at least 8 different gRNAs to at least 45 different gRNAs, atleast 8 different gRNAs to at least 40 different gRNAs, at least 8different gRNAs to at least 35 different gRNAs, 8 different gRNAs to atleast 30 different gRNAs, at least 8 different gRNAs to at least 25different gRNAs, 8 different gRNAs to at least 20 different gRNAs, atleast 8 different gRNAs to at least 16 different gRNAs, or 8 differentgRNAs to at least 12 different gRNAs.

The gRNA may comprise a complementary polynucleotide sequence of thetarget DNA sequence followed by a PAM sequence. The gRNA may comprise a“G” at the 5′ end of the complementary polynucleotide sequence. The gRNAmay comprise at least a 10 base pair, at least a 11 base pair, at leasta 12 base pair, at least a 13 base pair, at least a 14 base pair, atleast a 15 base pair, at least a 16 base pair, at least a 17 base pair,at least a 18 base pair, at least a 19 base pair, at least a 20 basepair, at least a 21 base pair, at least a 22 base pair, at least a 23base pair, at least a 24 base pair, at least a 25 base pair, at least a30 base pair, or at least a 35 base pair complementary polynucleotidesequence of the target DNA sequence followed by a PAM sequence. The PAMsequence may be “NGG”, where “N” can be any nucleotide. The gRNA maytarget at least one of the promoter region, the enhancer region or thetranscribed region of the target gene. The gRNA may include a nucleicacid sequence of at least one of SEQ ID NOs: sgRNA 1110-4:GCTTCTGTGTTGAAGACTCA (SEQ ID NO: 45), sgRNA 1110-6:GTAGCTGTGGGCCCACAACC(SEQ ID NO: 46), sgRNA Cc12-4: GCTCTTCCTCCACCACCATGC (SEQ ID NO: 47).

8. Multiplex CRISPR/Cas9-Based System

A multiplex CRISPR/Cas9-Based System which includes aCRISPR/CRISPR-associated (Cas) 9-based system, such as Cas9 or dCas9,and multiple gRNAs may be used to target one or more endogenous genes.This platform utilizes a convenient Golden Gate cloning method torapidly incorporate up to four independent sgRNA expression cassettesinto a single lentiviral vector. In some embodiments, the platform isincorporated into adeno-associated virus vectors or anintegrase-deficient lentivirus vector. Each sgRNA was efficientlyexpressed and could mediate multiplex gene editing at diverse loci inimmortalized and primary human cell lines.

The multiplex CRISPR/Cas9-Based System allows efficient multiplex geneediting for simultaneously inactivating multiple genes. The CRISPR/Cas9system can simultaneously target multiple distinct genomic loci byco-expressing a single Cas9 protein with two or more sgRNAs, making thissystem uniquely suited for multiplex gene editing or synergisticactivation applications. The CRISPR/Cas9 system greatly expedites theprocess of molecular targeting to new sites by simply modifying theexpressed sgRNA molecule. The single lentiviral vector may be combinedwith methods for achieving inducible control of these components, eitherby chemical or optogenetic regulation, to facilitate investigation ofthe dynamics of gene regulation in both time and space.

a) Modified Lentiviral Vector

The multiplex CRISPR/Cas9-based system includes a modified lentiviralvector. The modified lentiviral vector includes a first polynucleotidesequence encoding a Cas9 fusion protein and a second polynucleotidesequence encoding at least one sgRNA. The first polynucleotide sequencemay be operably linked to a promoter. The promoter may be a constitutivepromoter, an inducible promoter, a repressible promoter, or aregulatable promoter.

The second polynucleotide sequence encodes at least 1 sgRNA. Forexample, the second polynucleotide sequence may encode at least 1 sgRNA,at least 2 sgRNAs, at least 3 sgRNAs, at least 4 sgRNAs, at least 5sgRNAs, at least 6 sgRNAs, at least 7 sgRNAs, at least 8 sgRNAs, atleast 9 sgRNAs, at least 10 sgRNAs, at least 11 sgRNA, at least 12sgRNAs, at least 13 sgRNAs, at least 14 sgRNAs, at least 15 sgRNAs, atleast 16 sgRNAs, at least 17 sgRNAs, at least 18 sgRNAs, at least 19sgRNAs, at least 20 sgRNAs, at least 25 sgRNA, at least 30 sgRNAs, atleast 35 sgRNAs, at least 40 sgRNAs, at least 45 sgRNAs, or at least 50sgRNAs. The second polynucleotide sequence may encode between 1 sgRNAand 50 sgRNAs, between 1 sgRNA and 45 sgRNAs, between 1 sgRNA and 40sgRNAs, between 1 sgRNA and 35 sgRNAs, between 1 sgRNA and 30 sgRNAs,between 1 sgRNA and 25 different sgRNAs, between 1 sgRNA and 20 sgRNAs,between 1 sgRNA and 16 sgRNAs, between 1 sgRNA and 8 different sgRNAs,between 4 different sgRNAs and 50 different sgRNAs, between 4 differentsgRNAs and 45 different sgRNAs, between 4 different sgRNAs and 40different sgRNAs, between 4 different sgRNAs and 35 different sgRNAs,between 4 different sgRNAs and 30 different sgRNAs, between 4 differentsgRNAs and 25 different sgRNAs, between 4 different sgRNAs and 20different sgRNAs, between 4 different sgRNAs and 16 different sgRNAs,between 4 different sgRNAs and 8 different sgRNAs, between 8 differentsgRNAs and 50 different sgRNAs, between 8 different sgRNAs and 45different sgRNAs, between 8 different sgRNAs and 40 different sgRNAs,between 8 different sgRNAs and 35 different sgRNAs, between 8 differentsgRNAs and 30 different sgRNAs, between 8 different sgRNAs and 25different sgRNAs, between 8 different sgRNAs and 20 different sgRNAs,between 8 different sgRNAs and 16 different sgRNAs, between 16 differentsgRNAs and 50 different sgRNAs, between 16 different sgRNAs and 45different sgRNAs, between 16 different sgRNAs and 40 different sgRNAs,between 16 different sgRNAs and 35 different sgRNAs, between 16different sgRNAs and 30 different sgRNAs, between 16 different sgRNAsand 25 different sgRNAs, or between 16 different sgRNAs and 20 differentsgRNAs. Each of the polynucleotide sequences encoding the differentsgRNAs may be operably linked to a promoter. The promoters that areoperably linked to the different sgRNAs may be the same promoter. Thepromoters that are operably linked to the different sgRNAs may bedifferent promoters. The promoter may be a constitutive promoter, aninducible promoter, a repressible promoter, or a regulatable promoter.

At least one sgRNA may bind to a target gene or loci. If more than onesgRNA is included, each of the sgRNAs binds to a different target regionwithin one target loci or each of the sgRNA binds to a different targetregion within different gene loci. The fusion protein may include Cas9protein or iCas9-VP64 protein. The fusion protein may include a VP64domain, a p300 domain, or a KRAB domain.

b) Adeno-Associated Virus Vectors

AAV may be used to deliver CRISPRs to the cell using various constructconfigurations. For example, AAV may deliver Cas9 and gRNA expressioncassettes on separate vectors. Alternatively, if the small Cas9proteins, derived from species such as Staphylococcus aureus orNeisseria meningitidis, are used then both the Cas9 and up to two gRNAexpression cassettes may be combined in a single AAV vector within the4.7 kb packaging limit.

The composition, as described above, includes a modifiedadeno-associated virus (AAV) vector. The modified AAV vector may becapable of delivering and expressing the site-specific nuclease in thecell of a mammal. For example, the modified AAV vector may be anAAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy23:635-646). The modified AAV vector may be based on one or more ofseveral capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9.The modified AAV vector may be based on AAV2 pseudotype with alternativemuscle-tropic AAV capsids, such as AAV2/1, AAV2/6, AAV2/7, AAV2/8,AAV2/9, AAV2.5 and AAV/SASTG vectors that efficiently transduce skeletalmuscle or cardiac muscle by systemic and local delivery (Seto et al.Current Gene Therapy (2012) 12:139-151).

9. Methods of Generating the Modified Cell

Also provided herein is a method of generating the modified cell. Themethod comprises administering a CRISPR/Cas 9 system to the target cell,as described above. Use of the CRISPR/Cas 9 system to deliver thesite-specific nuclease to the cell may delete or replace the endogenousgene or fragment thereof thus generating the modified endogenous gene.The site-specific nuclease may be used to introduce site-specific doublestrand breaks at targeted genomic loci. Site-specific double-strandbreaks are created when the site-specific nuclease binds to a target DNAsequences, thereby permitting cleavage of the target DNA. This DNAcleavage may stimulate the natural DNA-repair machinery, leading to oneof two possible repair pathways: homology-directed repair (HDR) or thenon-homologous end joining (NHEJ) pathway.

The present disclosure is directed to generating the modified cell witha site-specific nuclease without a repair template. The disclosedsite-specific nucleases may involve using homology-directed repair ornuclease-mediated non-homologous end joining (NHEJ)-based correctionapproaches, which enable efficient correction in proliferation-limitedprimary cell lines that may not be amenable to homologous recombinationor selection-based gene correction. This strategy integrates the rapidand robust assembly of active site-specific nucleases with an efficientgene editing method for generating the modified cell. The method mayinvolve homology-directed repair or non-homologous end joining.

10. Methods of Treating a Disease

The present disclosure is directed to a method of treating a subject inneed thereof. The method comprises administering to subject thecomposition for cell therapy, as described above. The subject may havediseases include a variety of acute and chronic diseases including butnot limited to genetic, degenerative, or autoimmune diseases and obesityrelated conditions. Diseases include acute and chronic immune andautoimmune pathologies, such as, but not limited to, rheumatoidarthritis (RA), juvenile chronic arthritis (JCA), tissue ischemia,thyroiditis, graft versus host disease (GVHD), scleroderma, diabetesmellitus, Graves' disease, disc degeneration and low back pain, allergy,acute or chronic immune disease associated with an allogenictransplantation, such as, but not limited to, renal transplantation,cardiac transplantation, bone marrow transplantation, livertransplantation, pancreatic transplantation, small intestinetransplantation, lung transplantation and skin transplantation;infections, including, but not limited to, sepsis syndrome, cachexia,circulatory collapse and shock resulting from acute or chronic bacterialinfection, acute and chronic parasitic and/or infectious diseases,bacterial, viral or fungal, such as a human immunodeficiency virus(HIV), acquired immunodeficiency syndrome (AIDS) (including symptoms ofcachexia, autoimmune disorders, AIDS dementia complex and infections);inflammatory diseases, such as chronic inflammatory pathologies,including chronic inflammatory pathologies such as, but not limited to,sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis,osteogenesis imperfecta, and Crohn's pathology or disease; vascularinflammatory pathologies, such as, but not limited to, disseminatedintravascular coagulation, atherosclerosis, Kawasaki's pathology andvasculitis syndromes, such as, but not limited to, polyarteritis nodosa,Wegener's granulomatosis, Henoch-Schonlein purpura, giant cell arthritisand microscopic vasculitis of the kidneys; chronic active hepatitis;Sjogren's syndrome; spondyloarthropathies, such as ankylosingspondylitis, psoriatic arthritis and spondylitis, enteropathic arthritisand spondylitis, reactive arthritis and arthritis associated withinflammatory bowel disease; and uveitis; neurodegenerative diseases,including, but not limited to, demyelinating diseases, such as multiplesclerosis and acute transverse myelitis; myasthenia gravis;extrapyramidal and cerebellar disorders, such as lesions of thecorticospinal system; disorders of the basal ganglia or cerebellardisorders; hyperkinetic movement disorders, such as Huntington's choreaand senile chorea; drug-induced movement disorders, such as thoseinduced by drugs which block central nervous system (CNS) dopaminereceptors; hypokinetic movement disorders, such as Parkinson's disease;progressive supranuclear palsy; cerebellar and spinocerebellardisorders, such as astructural lesions of the cerebellum;spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia,cerebellar cortical degenerations, multiple systems degenerations(Mencel, Dejerine-Thomas, Shi-Drager, and MachadoJoseph)); and systemicdisorders (Refsum's disease, abetalipoprotienemia, ataxia,telangiectasia, and mitochondrial multisystem disorder); disorders ofthe motor unit, such as neurogenic muscular atrophies (anterior horncell degeneration, such as amyotrophic lateral sclerosis, infantilespinal muscular atrophy and juvenile spinal muscular atrophy);Alzheimer's disease; Down's syndrome in middle age; diffuse Lewy bodydisease; senile dementia of Lewy body type; Wernicke-Korsakoff syndrome;chronic alcoholism; primary biliary cirrhosis; cryptogenic fibrosingalveolitis and other fibrotic lung diseases; hemolytic anemia;Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis,Hallervorden-Spatz disease; and dementia pugilistica, or any subsetthereof; and malignant pathologies involving TNF-secreting tumors orother malignancies involving TNF, such as, but not limited to, leukemias(acute, chronic myelocytic, chronic lymphocytic and/or myelodyspasticsyndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such asmalignant lymphomas (Burkitt's lymphoma or Mycosis fungoides)).

i) Chronic Inflammatory Disease

Chronic inflammatory diseases such as arthritis are characterized byaberrant activity of cytokines such as tumor necrosis factor-a (TNF-a)and interleukin-1 (IL-1). These pro-inflammatory mediators are expressedby a wide variety of cells in musculoskeletal tissues, includingmyotubes, satellite cells, chondrocytes, synovial fibroblasts,osteoblasts, and resident as well as infiltrating innate immune cells.These cell types are also capable of responding to TNF-a and IL-1 athrough canonical signaling via their cognate cell surface receptors. Inhealthy tissue, appropriate signaling of TNF-a and IL-1 contributes toorgan and tissue homeostasis. In this state, these mediators promotetissue remodeling, orchestrate phagocytosis of cellular debris andimmunogenic substrates, and coordinate transitions between niche stemcell quiescence and proliferation/differentiation programs. TNF-a hasalso been shown to enhance stem cell differentiation in a variety ofefforts to enhance MSC osteogenesis. However, in chronic diseases,elevated levels of these pro-inflammatory cytokines can lead directly topain, cytotoxicity, accelerated tissue catabolism or wasting, andexhaustion of resident stem cell niches.

A regenerative medicine approach may be used to treat chronicinflammatory diseases by generating custom-designed cells that canexecute real-time, programmed responses to environmental cues, includingpro-inflammatory cytokines. Modified cells, such as stem cells, may begenerated with the ability to antagonize IL-1- and TNFa-mediatedinflammation in an auto-regulated, feedback-controlled manner. Targetedgene addition of IL-1 and TNF antagonists may be performed at the Ccl2locus to confer cytokine-activated and feedback-controlled expression ofbiologic therapies. Genome-edited stem cells may be used to engineerarticular cartilage tissue to establish the efficacy of self-regulatedtherapy toward protection of tissues against cytokine-induceddegeneration. This approach of repurposing normally degradativesignaling pathways may facilitate transient production of cytokineantagonists and permit effective treatment of chronic diseases whileovercoming limitations associated with delivery of large drug doses orconstitutive overexpression of biologic compounds.

A therapeutic molecule may be any number of exogenous anti-cytokinetherapies that effectively counteract the negative sequelae of TNF-a andIL-1 dysregulation. For example, therapeutic molecules may includecompetitive antagonists such as IL-1 receptor antagonist (IL-1Ra,anakinra), which alleviate symptoms of rheumatoid arthritis and theonset of post-traumatic arthritis; anti-TNF therapies, such as thesoluble type 2 TNF receptor (etanercept) and monoclonal antibodies toTNF-a (adalimumab, infliximab), which have demonstrated efficacy towardoffsetting pain associated with chronic and rheumatic diseases,including arthritis, ankylosing spondylitis, Crohn disease, plaquepsoriasis, and ulcerative colitis; type I soluble TNFR receptor(sTNFR1), which generally provided in the context of relatively high orunregulated doses.

11. Methods of Cancer Therapy

The compositions may be used in methods of cancer therapy where theimmune system is used to treat cancer. Immunotherapies fall into threemain groups: cellular, antibody and cytokine. They exploit the fact thatcancer cells often have subtly different molecules on their surface thatcan be detected by the immune system. These molecules, known as cancerantigens, are most commonly proteins, but also include molecules such ascarbohydrates. Immunotherapy is used to provoke the immune system intoattacking the tumor cells by using these antigens as targets.

The compositions may be used in cellular therapies, also known as cancervaccines, usually involve the removal of immune cells from the blood orfrom a tumor. Immune cells specific for the tumor may be modified,cultured and returned to the patient where the immune cells attack thecancer. Cell types that can be used in this way are natural killercells, lymphokine-activated killer cells, cytotoxic T cells anddendritic cells.

Interleukin-2 and interferon-a are examples of cytokines, proteins thatregulate and coordinate the behavior of the immune system. They have theability to enhance anti-tumor activity and thus can be used as cancertreatments. Interferon-a is used in the treatment of hairy-cellleukemia, AIDS-related Kaposi's sarcoma, follicular lymphoma, chronicmyeloid leukemia and malignant melanoma. Interleukin-2 is used in thetreatment of malignant melanoma and renal cell carcinoma.

Dendritic cell therapy provokes anti-tumor responses by causingdendritic cells to present tumor antigens. Dendritic cells presentantigens to lymphocytes, which activates them, priming them to killother cells that present the antigen. In cancer treatment they aidcancer antigen targeting. One method of inducing dendritic cells topresent tumor antigens is by vaccination with short peptides (smallparts of protein that correspond to the protein antigens on cancercells). These peptides on their own do not stimulate a strong immuneresponse and may be given in combination with adjuvants (highlyimmunogenic substances). This provokes a strong response, while alsoproducing a (sometimes) robust anti-tumor response by the immune system.Other adjuvants include proteins or other chemicals that attract and/oractivate dendritic cells, such as granulocyte macrophagecolony-stimulating factor (GM-CSF). Dendritic cells can also beactivated within the body (in vivo) by making tumor cells express(GM-CSF). This can be achieved by either genetically engineering tumorcells that produce GM-CSF or by infecting tumor cells with an oncolyticvirus that expresses GM-CSF.

Another strategy is to remove dendritic cells from the blood of apatient and activate them outside the body (ex vivo). The dendriticcells are activated in the presence of tumor antigens, which may be asingle tumor-specific peptide/protein or a tumor cell lysate (a solutionof broken down tumor cells). These activated dendritic cells are putback into the body where they provoke an immune response to the cancercells. Adjuvants are sometimes used systemically to increase theanti-tumor response provided by ex vivo activated dendritic cells. Moremodern dendritic cell therapies include the use of antibodies that bindto receptors on the surface of dendritic cells. Antigens can be added tothe antibody and can induce the dendritic cells to mature and provideimmunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8or CD40 have been used as targets by antibodies to produce immuneresponses.

Cytokines are a broad group of proteins produced by many types of cellspresent within a tumor. They have the ability to modulate immuneresponses. The tumor often employs it to allow it to grow and manipulatethe immune response. These immune-modulating effects allow them to beused as drugs to provoke an immune response. Two commonly used groups ofcytokines are interferons and interleukins.

Interferons are cytokines produced by the immune system. They areusually involved in anti-viral response, but also have use for cancer.The three groups of interferons (IFNs) are type I (IFNa and IFN(3), typeII (IFNy) and type III (IFNX). IFNa has been approved for use inhairy-cell leukemia, AIDS-related Kaposi's sarcoma, follicular lymphoma,chronic myeloid leukemia and melanoma. Type I and II IFNs have beenresearched extensively and although both types promote anti-tumor immunesystem effects, only type I IFNs have been shown to be clinicallyeffective. IFN2 shows promise for its anti-tumor effects in animalmodels.

Interleukins are a group of cytokines with a wide array of immune systemeffects. Interleukin-2 is used in the treatment of malignant melanomaand renal cell carcinoma. In normal physiology it promotes both effectorT cells and T-regulatory cells, but its exact mechanism in the treatmentof cancer is unknown.

12. Methods of Regenerative Therapy Using Cell Therapy

Regenerative medicine provides the exciting potential for cell-basedtherapies to treat many diseases and restore damaged tissues usingengineered cells for musculoskeletal applications. Modified cellsderived from a myriad of adult tissues and differentiated down a lineageof choice may be tailored at the scale of the genome withapplication-dependent features. The compositions described here havebroad applicability in regenerative medicine. For example, the abilityto immobilize gene delivery vehicles capable of dictating cell fate andorchestrating ECM deposition may allow future investigators to controlthe spatial patterning of tissue development. This approach could indeedbe applied toward engineering tissues comprised of multiple cell typesand organized into regions of varied and distinct ECM constituents, apersistent challenge in the field of orthopaedic tissue engineering.Furthermore, diseases may be treated that involve complex interactionsbetween multiple organ systems, those that drive deterioration oftissues that are not amenable to total replacement, or those in whichdiscrimination between pathologic and healthy tissue/cells may be subtleor require real-time determination for safe and effective alleviation ofdisease. Such conditions may be most efficiently addressed by cells thatcan infiltrate, intelligently detect dysfunction, and deploy predefinedtherapeutic programs to resolve anomalous behavior of endogenous cellsor ECM disorganization/degeneration. Employing these and other toolsfrom synthetic biology together under the auspices of a functionalcellular and tissue engineering paradigm, which aims to fullycharacterize and recapitulate features critical for successfulcell/tissue replacement, will likely serve to advance the field ofregenerative medicine toward the establishment of clinically effectivetherapies for a host of diseases.

In some embodiments, the site-specific nucleases may be used to generatefunctional deficiencies or complete knock-out of the proteins coded bythe targeted genes in human iPSCs. Genetically modified iPSCs may bedifferentiated into chondrocytes using established techniques.Feedback-controlled gene circuits may be designed to modulate theproduction of soluble TNF receptors—specifically soluble TNF receptor 1(sTNFR1), which blocks TNF signaling—in response to dynamic TNF levels.In some embodiments, this process may be performed in inducedpluripotent stem cells (iPSCs), which can be expanded indefinitely, thusfacilitating the complex genetic manipulations required for genomeediting. To produce an implant with long-term in vivo stability, therewired iPSCs may be differentiated into cartilage cells (chondrocytes),a robust, non-migratory cell that naturally responds to TNF. These cellsmay be formed into a tissue-engineered cartilage implant (see FIG. 1)that can be implanted in the joint to repair damaged cartilage orsubcutaneously to provide self-regulated, systemic anti-TNF.

a) Osteoarthritis

The modified cells may be used in musculoskeletal regenerative medicineapplications, such as developing therapies for osteoarthritis.Osteoarthritis (OA) is a progressive disease of synovial jointscharacterized by the destruction of articular cartilage. Surgicaltreatment options for focal cartilage defects include arthroscopicdebridement, marrow stimulation via microfracture, and autologoustransplantation of host tissue or ex vivo expanded autologouschondrocytes. Most of these surgical options lead to the development ofa fibrocartilage matrix that serves only as a temporary solution to acomplex and demanding biomechanical problem. For larger defects, jointarthroplasty serves as the most promising treatment option. Whileeffective at restoring function to the joint, the need to revise anincreasing number of primary arthroplasties means that a morefunctional, long-term solution is needed.

Inflammation plays a key role in the pathogenesis and progression ofosteoarthritis (OA) and may compromise engineered tissue substitutes.Chondrocytes and synovial fibroblasts in OA joints are subjected toincreased interleukin (IL)-1, 1L-6, 1L-17 and tumor necrosis factor(TNF)-a signaling. The activity of these cytokines in OA joints leads toincreased production of matrix metalloproteinases (MMPs), aggrecanases,inducible nitric oxide synthase, and prostaglandin E2. These and otherfactors ultimately lead to suppression of cartilage-specific genes suchas COL2A1, downregulation of proteoglycan levels, degeneration ofextracellular matrix, and chondrocyte apoptosis. Furthermore, prolongedinflammatory signaling mediated by IL-la inhibits chondrogenic inductionof stem cells and results in degradation of stem cell derived cartilage.The pro-inflammatory environment of the OA joint therefore necessitatesa tissue substitute designed to resist inflammation-mediateddegradation.

13. Target Cells

The target cells that are used to generate the modified cells may bestem cells, such as embryonic stem cells (ES) and adult stem cells(somatic stem cells or tissue-specific stem cells), induced pluripotentstem cells (iPSCs), progenitor cells, fibroblasts, cardiomyocytes,hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroidleukemia cell line, bone cells, synovial cells, tendon cells, ligamentcells, meniscus cells, adipose cells, B-cells, dendritic cells, naturalkiller cells, or T-cells.

a) Embryonic Stem Cells (ES)

(ES) cells are isolated from the inner cell mass of blastocysts ofpreimplantation-stage embryos. These cells require specific signals todifferentiate to the desired cell type; if simply injected directly,they will differentiate into many different types of cells, resulting ina tumor derived from this abnormal pluripotent cell development (ateratoma). The directed differentiation of ES cells and avoidance oftransplant rejection are just two of the hurdles that ES cellresearchers still face. With their potential for unlimited expansion andpluripotency, ES cells are a potential source for regenerative medicineand tissue replacement after injury or disease.

b) Adult Stem Cells

Adult stem cells are undifferentiated cells, found throughout the bodyafter development, that multiply by cell division to replenish dyingcells and regenerate damaged tissues. Also known as somatic stem cells,they can be found in juvenile as well as adult animals and human bodies.Scientific interest in adult stem cells is centered on their ability todivide or self-renew indefinitely, and generate all the cell types ofthe organ from which they originate, potentially regenerating the entireorgan from a few cells. Unlike embryonic stem cells, the use of humanadult stem cells in research and therapy is not considered to becontroversial, as they are derived from adult tissue samples rather thanhuman 5 day old embryos generated by IVF (in vitro fertility) clinicsdesignated for scientific research. They have mainly been studied inhumans and model organisms such as mice and rat.

The production of adult stem cells does not require the destruction ofan embryo. Additionally, when adult stem cells are obtained from theintended recipient (an autograft) there is no risk of immune rejection.Adult stem cell treatments have been successfully used for many years totreat leukemia and related bone/blood cancers through bone marrowtransplants.

i) Hematopoietic Stem Cells

Hematopoietic stem cells are found in the bone marrow and umbilical cordblood and give rise to all the blood cell types.

ii) Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) are of stromal origin and maydifferentiate into a variety of tissues and cell types, including:osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes(muscle cells) adipocytes (fat cells). MSCs have been isolated fromplacenta, adipose tissue, lung, bone marrow and blood, Wharton's jellyfrom the umbilical cord and teeth (perivascular niche of dental pulp andperiodontal ligament). MSCs are attractive for clinical therapy due totheir ability to differentiate, provide trophic support, and modulateinnate immune response.

iii) Endothelial Stem Cells

Endothelial stem cells are one of the three types of multipotent stemcells found in the bone marrow. They are a rare and controversial groupwith the ability to differentiate into endothelial cells, the cells thatline blood vessels.

iv) Epithelial Stem Cells

Self-renewing tissues, such as the epidermis and hair follicle,continuously generate new cells to replenish the dead squames and hairs,which are sloughed into the environment. Therefore, perhaps the simplestdefinition of an epithelial stem cell is based on lineage: a stem cellis the cell of origin for terminally differentiated cells in adulttissues. For example, tracing the lineage of a corneocyte or hair cellback to its ultimate source in the adult skin leads to a stem cell.However, because the tools required to perform lineage analysis have notbeen available until recently, investigators have principally adopteddefinitions from the hematopoietic system. In particular, stem cellswere felt to be self-renewing, multipotent, and clonogenic, similar tostem cells in the hematopoietic system that can regenerate all of theblood lineages from one cell after transplantation. In contrast to thehematopoietic stem cell field, cutaneous epithelial stem cell biologistsalso relied heavily on quiescence as a major stem cell characteristic.

v) Neural Stem Cells

The existence of stem cells in the adult brain has been postulatedfollowing the discovery that the process of neurogenesis, the birth ofnew neurons, continues into adulthood in rats. The presence of stemcells in the mature primate brain was first reported in 1967. It hassince been shown that new neurons are generated in adult mice, songbirdsand primates, including humans. Normally, adult neurogenesis isrestricted to two areas of the brain—the subventricular zone, whichlines the lateral ventricles, and the dentate gyrus of the hippocampalformation. Although the generation of new neurons in the hippocampus iswell established, the presence of true self-renewing stem cells therehas been debated. Under certain circumstances, such as following tissuedamage in ischemia, neurogenesis can be induced in other brain regions,including the neocortex.

Neural stem cells are commonly cultured in vitro as so calledneurospheres—floating heterogeneous aggregates of cells, containing alarge proportion of stem cells. They can be propagated for extendedperiods of time and differentiated into both neuronal and glia cells,and therefore behave as stem cells. However, some recent studies suggestthat this behavior is induced by the culture conditions in progenitorcells, the progeny of stem cell division that normally undergo astrictly limited number of replication cycles in vivo. Furthermore,neurosphere-derived cells do not behave as stem cells when transplantedback into the brain.

Neural stem cells share many properties with hematopoietic stem cells(HSCs). Remarkably, when injected into the blood, neurosphere-derivedcells differentiate into various cell types of the immune system.

vi) Mammary Stem Cells

Mammary stem cells provide the source of cells for growth of the mammarygland during puberty and gestation and play an important role incarcinogenesis of the breast. Mammary stem cells have been isolated fromhuman and mouse tissue as well as from cell lines derived from themammary gland. Single such cells can give rise to both the luminal andmyoepithelial cell types of the gland, and have been shown to have theability to regenerate the entire organ in mice.

vii) Intestinal Stem Cells

Intestinal stem cells divide continuously throughout life and use acomplex genetic program to produce the cells lining the surface of thesmall and large intestines. Intestinal stem cells reside near the baseof the stem cell niche, called the crypts of Lieberkuhn. Intestinal stemcells are probably the source of most cancers of the small intestine andcolon.

viii) Olfactory Adult Stem Cells

Olfactory adult stem cells have been successfully harvested from thehuman olfactory mucosa cells, which are found in the lining of the noseand are involved in the sense of smell. If they are given the rightchemical environment these cells have the same ability as embryonic stemcells to develop into many different cell types. Olfactory stem cellshold the potential for therapeutic applications and, in contrast toneural stem cells, can be harvested with case without harm to thepatient. This means they can be easily obtained from all individuals,including older patients who might be most in need of stem celltherapies.

ix) Neural Crest Stem Cells

Hair follicles contain two types of stem cells, one of which appears torepresent a remnant of the stem cells of the embryonic neural crest.Similar cells have been found in the gastrointestinal tract, sciaticnerve, cardiac outflow tract and spinal and sympathetic ganglia. Thesecells can generate neurons, Schwann cells, myofibroblast, chondrocytesand melanocytes.

x) Testicular Cells

Multipotent stem cells with a claimed equivalency to embryonic stemcells have been derived from spermatogonial progenitor cells found inthe testicles of laboratory mice. The extracted stem cells are known ashuman adult germ line biggmacc stem cells (GSCs). Multipotent stem cellshave also been derived from germ cells found in human testicles.

c) Induced Stem Cells

Induced stem cells (iSC) are stem cells artificially derived fromsomatic, reproductive, pluripotent or other cell types by deliberateepigenetic reprogramming. They are classified as totipotent (iTC),pluripotent (iPSC) or progenitor (multipotent-iMSC, also called aninduced multipotent progenitor cell-iMPC) or unipotent (iUSC) accordingto their developmental potential and degree of dedifferentiation.

iPSCs are somatic cells that have been genetically reprogrammed to anembryonic stem cell—like state by being forced to express genesimportant for maintaining the defining properties of embryonic stemcells. Although additional research is needed, iPSCs are already usefultools for drug development and modeling of diseases, and scientists hopeto use them in transplantation medicine. In addition, tissues derivedfrom iPSCs will be a nearly identical match to the cell donor and thusprobably avoid rejection by the immune system. By studying iPSCs andother types of pluripotent stem cells, researchers may learn how toreprogram cells to repair damaged tissues in the human body.

xi) Lung and Airway Epithelial Cells

Chronic lung diseases such as idiopathic pulmonary fibrosis and cysticfibrosis or chronic obstructive pulmonary disease and asthma are leadingcauses of morbidity and mortality worldwide with a considerable human,societal, and financial burden. Several protocols have been developedfor generation of the most cell types of the respiratory system, whichmay be useful for deriving patient-specific therapeutic cells.

xii) Reproductive Cells

Some lines of iPSCs have the potentiality to differentiate into malegerm cells and oocyte-like cells in an appropriate niche (by culturingin retinoic acid and porcine follicular fluid differentiation medium orseminiferous tubule transplantation). Moreover, iPSC transplantationmake a contribution to repairing the testis of infertile mice,demonstrating the potentiality of gamete derivation from iPSCs in vivoand in vitro.

d) T-Cells

T cells are a type of lymphocyte (in turn, a type of white blood cell)that play a central role in cell-mediated immunity. They can bedistinguished from other lymphocytes, such as B cells and natural killercells (NK cells), by the presence of a T-cell receptor (TCR) on the cellsurface. They are called T cells because they mature in the thymus(although some also mature in the tonsils). The several subsets of Tcells each have a distinct function. The majority of human T cellsrearranges their alpha/beta T cell receptors and are termed alpha beta Tcells and are part of adaptive immune system. Specialized gamma delta Tcells, which comprise a minority of T cells in the human body (morefrequent in ruminants), have invariant TCR (with limited diversity), caneffectively present antigens to other T cells and are considered to bepart of the innate immune system.

The T cell includes any of a CD8-positive T cell (cytotoxic T cell:CTL), a CD4-positive T cell (helper T cell), a suppressor T cell, aregulatory T cell such as a controlling T cell, an effector cell, anaive T cell, a memory T cell, an al3T cell expressing TCR a and Pchains, and a y6T cell expressing TCR y and 6 chains. The T cellincludes a precursor cell of a T cell in which differentiation into a Tcell is directed. Examples of “cell populations containing T cells”include, in addition to body fluids such as blood (peripheral blood,umbilical blood etc.) and bone marrow fluids, cell populationscontaining peripheral blood mononuclear cells (PBMC), hematopoieticcells, hematopoietic stem cells, umbilical blood mononuclear cells etc.,which have been collected, isolated, purified or induced from the bodyfluids. Further, a variety of cell populations containing T cells andderived from hematopoietic cells can be used in the present invention.These cells may have been activated by cytokine such as 1L-2 in vivo orex vivo. As these cells, any of cells collected from a living body, orcells obtained via ex vivo culture, for example, a T cell populationobtained by the method of the present invention as it is, or obtained byfreeze preservation, can be used.

e) Chimeric Antigen Receptor T-Cells (CAR-T)

Artificial T cell receptors (also known as chimeric T cell receptors,chimeric immunoreceptors, chimeric antigen receptors (CARs)) areengineered receptors, which graft an arbitrary specificity onto animmune effector cell. These receptors may be used to graft thespecificity of a monoclonal antibody onto a T cell. CARs may consist ofa monoclonal antibody fragment, such as a single-chain variable fragment(scFv), that presents on the outside of T-cell membranes, and is fusedto intracellularly-facing stimulatory molecules. The scFv portion mayrecognize the tumor target. Upon binding, the intracellular stimulatoryportions may initiate a signal to activate the T cell.

Artificial T cell receptors may be used as therapy for cancer usingadoptive cell transfer. T cells are removed from a patient and modifiedso that they express receptors specific to the particular form ofcancer. The T cells, which can then recognize and kill the cancer cells,are reintroduced into the patient. Modification of T-cells sourced fromdonors other than the patient may also be used.

14. Methods of Delivery

Provided herein is a method for delivering the modified cells. Themodified cells may be injected or implanted into a mammal, usedexogenously, or developed into tissue engineered constructs. The mammalmay be human, non-human primate, cow, pig, sheep, goat, antelope, bison,water buffalo, bovids, deer, hedgehogs, elephants, llama, alpaca, mice,rats, or chicken, and preferably human, cow, pig, or chicken.

Also, provided herein is a method for delivering the CRISPR/Cas9-basedsystem to the target cell. The delivery of the CRISPR/Cas9-based systemmay be the transfection or electroporation of the CRISPR/Cas9-basedsystem as a nucleic acid molecule that is expressed in the cell anddelivered to the surface of the cell. The nucleic acid molecules may beelectroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector libdevices. Several different buffers may be used, including BioRadelectroporation solution, Sigma phosphate-buffered saline product #D8537(PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V(N.V.). Transfections may include a transfection reagent, such asLipofectamine 2000. Upon delivery of the CRISPR/Cas9 system to thetissue, and thereupon the vector into the cells of the mammal, thetransfected cells will express the CRISPR/Cas9-based system and/or asite-specific nuclease.

15. Constructs and Plasmids

A genetic construct, such as a plasmid, may comprise a nucleic acid thatencodes the CRISPR/Cas9-based system, such as the Cas9 protein and Cas9fusion proteins and/or at least one of the gRNAs. The genetic constructsmay encode a modified AAV vector and a nucleic acid sequence thatencodes the site-specific nuclease, as disclosed herein. The geneticconstruct, such as a plasmid, may comprise a nucleic acid that encodesthe site-specific nuclease. The genetic constructs may encode a modifiedlentiviral vector, as disclosed herein. The genetic construct, such as aplasmid, may comprise a nucleic acid that encodes the Cas9-fusionprotein and at least one sgRNA. The genetic construct may be present inthe cell as a functioning extrachromosomal molecule. The geneticconstruct may be a linear minichromosome including centromere, telomeresor plasmids or cosmids.

The genetic construct may also be part of a genome of a recombinantviral vector, including recombinant lentivirus, recombinant adenovirus,and recombinant adenovirus associated virus. The genetic construct maybe part of the genetic material in attenuated live microorganisms orrecombinant microbial vectors which live in cells. The geneticconstructs may comprise regulatory elements for gene expression of thecoding sequences of the nucleic acid. The regulatory elements may be apromoter, an enhancer, an initiation codon, a stop codon, or apolyadenylation signal.

The nucleic acid sequences may make up a genetic construct that may be avector. The vector may be capable of expressing the fusion protein, suchas the Cas9-fusion protein or site-specific nuclease, in the cell of amammal. The vector may be recombinant. The vector may compriseheterologous nucleic acid encoding the fusion protein, such as theCas9-fusion protein or site-specific nuclease. The vector may be aplasmid. The vector may be useful for transfecting cells with nucleicacid encoding the Cas9-fusion protein or site-specific nuclease, whichthe transformed host cell is cultured and maintained under conditionswherein expression of the Cas9-fusion protein or the site-specificnuclease system takes place.

Coding sequences may be optimized for stability and high levels ofexpression. In some instances, codons are selected to reduce secondarystructure formation of the RNA such as that formed due to intramolecularbonding.

The vector may comprise heterologous nucleic acid encoding theCRISPR/Cas9-based system or the site-specific nuclease and may furthercomprise an initiation codon, which may be upstream of theCRISPR/Cas9-based system or the site-specific nuclease coding sequence,and a stop codon, which may be downstream of the CRISPR/Cas9-basedsystem or the site-specific nuclease coding sequence. The initiation andtermination codon may be in frame with the CRISPR/Cas9-based system orthe site-specific nuclease coding sequence. The vector may also comprisea promoter that is operably linked to the CRISPR/Cas9-based system orthe site-specific nuclease coding sequence. The promoter operably linkedto the CRISPR/Cas9-based system or the site-specific nuclease codingsequence may be a promoter from simian virus 40 (SV40), a mouse mammarytumor virus (MMTV) promoter, a human immunodeficiency virus (HIV)promoter such as the bovine immunodeficiency virus (BIV) long terminalrepeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus(ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMVimmediate early promoter, Epstein Barr virus (EBV) promoter, or a Roussarcoma virus (RSV) promoter. The promoter may also be a promoter from ahuman gene such as human ubiquitin C (hUbC), human actin, human myosin,human hemoglobin, human muscle creatine, or human metalothionein. Thepromoter may also be a tissue specific promoter, such as a muscle orskin specific promoter, natural or synthetic. Examples of such promotersare described in US Patent Application Publication No. US20040175727,the contents of which are incorporated herein in its entirety.

The vector may also comprise a polyadenylation signal, which may bedownstream of the CRISPR/Cas9-based system or the site-specificnuclease. The polyadenylation signal may be a SV40 polyadenylationsignal, LTR polyadenylation signal, bovine growth hormone (bGH)polyadenylation signal, human growth hormone (hGH) polyadenylationsignal, or human J3-globin polyadenylation signal. The SV40polyadenylation signal may be a polyadenylation signal from a pCEP4vector (Invitrogen, San Diego, Calif.).

The vector may also comprise an enhancer upstream of theCRISPR/Cas9-based system, i.e., the Cas9 protein or Cas9 fusion proteincoding sequence or sgRNAs, or the site-specific nuclease. The enhancermay be necessary for DNA expression. The enhancer may be human actin,human myosin, human hemoglobin, human muscle creatine or a viralenhancer such as one from CMV, HA, RSV or EBV. Polynucleotide functionenhancers are described in U.S. Pat. Nos. 5,593,972, 5,962,428, andWO94/016737, the contents of each are fully incorporated by reference.The vector may also comprise a mammalian origin of replication in orderto maintain the vector extrachromosomally and produce multiple copies ofthe vector in a cell. The vector may also comprise a regulatorysequence, which may be well suited for gene expression in a mammalian orhuman cell into which the vector is administered. The vector may alsocomprise a reporter gene, such as green fluorescent protein (“GFP”)and/or a selectable marker, such as hygromycin (“Hygro”).

The vector may be expression vectors or systems to produce protein byroutine techniques and readily available starting materials includingSambrook et al., Molecular Cloning and Laboratory Manual, Second Ed.,Cold Spring Harbor (1989), which is incorporated fully by reference. Insome embodiments the vector may comprise the nucleic acid sequenceencoding the CRISPR/Cas9-based system, including the nucleic acidsequence encoding the Cas9 protein or Cas9 fusion protein and thenucleic acid sequence encoding at least one gRNA comprising the nucleicacid sequence of at least one of SEQ ID NOs: 45-47.

16. Kits

Provided herein is a kit, which may be used to generate the modifiedcell. The kit comprises a composition for generating the modified cell,as described above, and instructions for using said composition.Instructions included in kits may be affixed to packaging material ormay be included as a package insert. While the instructions aretypically written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this disclosure. Such media include, but arenot limited to, electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. As usedherein, the term “instructions” may include the address of an internetsite that provides the instructions.

The composition for generating the modified cell may include a modifiedAAV vector and a nucleotide sequence encoding a site-specific nuclease,as described above. The site-specific nuclease may include a ZFN, aTALEN, or CRISPR/Cas9-based system, as described above, thatspecifically binds and cleaves a mutated gene. The site-specificnuclease, as described above, may be included in the kit to specificallybind and target a particular region in the endogenous gene. Thesite-specific nuclease may be specific for an endogenous Uri or Ccl2gene, as described above. The kit may further include donor DNA, a gRNA,or a transgene, as described above.

At least one component may include at least one CRISPR/Cas9-basedsystem, as described above, which specifically targets a gene. The kitmay include a Cas9 protein or Cas9 fusion protein, a nucleotide sequenceencoding said Cas9 protein or Cas9 fusion protein, and/or at least onegRNA. The CRISPR/Cas9-based system, as described above, may be includedin the kit to specifically bind and target a particular target regionupstream, within or downstream of the coding region of the target gene.For example, a CRISPR/Cas9-based system may be specific for a promoterregion of a target gene or a CRISPR/Cas9-based system may be specificfor the coding region, as described above. The kit may include donorDNA, as described above.

17. Examples

The foregoing may be better understood by reference to the followingexamples, which are presented for purposes of illustration and are notintended to limit the scope of the invention. The present invention hasmultiple aspects, illustrated by the following non-limiting examples.

Example 1 Methods—Illrl

Induced pluripotent stem cells (iPSCs) with specific genomicmodifications were engineered with specific genomic alterations thatconfer resistance to inflammation, e.g., resistance to IL-1, and tovalidate the potential therapeutic utility of these designer stem cellsas a source for cartilage tissue engineering and regenerative medicine.CRISPR/Cas9 nucleases capable of mediating deletion of the signalpeptide sequence of the interleukin 1 receptor 1 gene (Illrl), theligand-binding receptor responsible for IL-la recognition and involvedfor IL-1 signal transduction, were transfected into murine iPSCs togenerate murine iPSCs deficient in Illrl. The ability of the cells tosynthesize a cartilaginous extracellular matrix (ECM) and resist theinflammation-mediated catabolism initiated by an IL-la assault wasevaluated. Clones were subsequently isolated. Three of 41 iPSC clonespossessed the Illrl+/− genotype and four possessed the Illrl−/−genotype. Flow cytometry confirmed Ill rl loss in Ill rl−/− genotypedcells. IL-la induced NF-KB transcriptional activity in Illrl+/+ andIllrl+/− cells but failed to do so in Illrl−/− cells. Cartilageengineered from Illrl−/− clones was resistant to IL-la-mediateddegradation, as indicated by gene expression analyses and thepreservation of sulfated glycosaminoglycan in the cartilageextracellular matrix, while cartilage derived from Illrl+/+ and Illrl+/−clones demonstrated a significant degradative response to theIL-1-mediated in vitro model of OA, including loss of more than 65% ofsGAG compared to controls. Using targeted gene editing nucleases,IL-1-resistant pluripotent cells were engineered, demonstrating thatstem cells can be tailored at the genomic scale with features suitablefor tissue engineering and regenerative medicine applications.

Induced Pluripotent Stem Cell Derivation and Culture. Murine inducedpluripotent stem cells were derived and cultured as previously described(Diekman et al., (2012) Proc. Natl. Acad. Sci. 109:19172-19177).Briefly, tail fibroblasts from adult C57BL/6 mice were transduced with alentiviral vector driving doxycycline-inducible expression of Oct4(Pou5f1), Sox2, Klf4, and c-myc (Carey et al., (2009) Proc. Natl. Acad.Sci. 106:157-162). Pluripotent cells were maintained on mitomycinC-treated mouse embryonic feeders (MEFs; Millipore) in medium comprisedof high glucose Dulbecco's modified Eagle's medium (DMEM) supplementedwith L-glutamine, sodium pyruvate, 20% fetal bovine serum, 100 nMminimum essential medium non-essential amino acids (NEAA; Gibco),551.tM13-mercaptoethanol (2-ME; Gibco), and 1,000 units of leukemiainhibitory factor (L1F; Millipore). A Col2a1-GFP reporter construct(Grant et al., (2000) Developmental Dynamics 218:394-400) wastransfected into cells by Nucleofection, and a clone stably expressingthe reporter upon chondrogenic induction was isolated after G418selection.

Genome Editing and Clonal Isolation. A plasmid encoding human codonoptimized Streptococcus pyogenes Cas9 (hCas9) was as previouslydescribed (Mali et al., (2013) Science 339:823-826) (Addgene plasmid#41815). Target sequences flanking exon 2 of Illrl and corresponding to5′-GCTTCTGTGTTGAAGACTCA-3′ (SEQ ID NO: 1) and 5′-GTAGCTGTGGGCCCACAACC-3′(SEQ ID NO: 2) were selected to generate the deletion of the Illrlsignal peptide sequence. To produce single chimeric guide RNA (sgRNA)expression vectors, complementary oligonucleotides containing each ofthe target sequences were hybridized, phosphorylated, and cloned into anexpression vector (Perez-Pinera et al., (2013) Nat. Meth. 10:973-976)(Addgene plasmid #47108) employing an human U6 promoter to driveexpression of a chimeric Streptococcus pyogenes crRNA/tracrRNA sequence.The gRNA sequences were sgRNA Illr1-4: GCTTCTGTGTTGAAGACTCA (SEQ ID NO:43) and sgRNA Illr1-6: GTAGCTGTGGGCCCACAACC (SEQ ID NO: 44). Prior totransfection, iPSCs were trypsinized and subjected to a 30-minute feedersubtraction. Lipofectamine 2000 (Life Technologies) was used followingmanufacturer's instructions to co-transfect 400 ng of each sgRNA and 800ng hCas9 into 100,000 iPSCs freshly plated on MEFs in complete,antibiotic-free iPSC medium in a 24-well plate. Cells were thensub-cultured on MEFs prior to single-cell deposition. In preparation forsingle cell deposition, iPSCs were feeder subtracted prior to overnightculture on 0.1% gelatin. Cells were then trypsinized and subjected to afinal feeder subtraction and then suspended in calcium- andmagnesium-free PBS, 1 mM EDTA, 25 mM HEPES, and 1% FBS. Individual cellswere then deposited into gelatin-coated wells of a 96-well plate. Cloneswere sub-cultured on gelatin for one additional passage to allow forscreening for the appropriate deletion via genomic PCR using thefollowing primer pair: Illrl detF—5′-TCATCTCCTGGTTAGTTATGGTATC-3′ (SEQID NO: 3) and Illrl detR—5′-CCGAGGCCAATGAGATTAAG-3′ (SEQ ID NO: 4). Asubset of each clone was lysed using QuickExtract (Epicentre) accordingto the manufacturer's instructions. The cell lysate was then diluted8-10 fold prior to use as template in a PCR using Q5 polymerase (NEB)according to manufacturer's instruction with the following cyclingparameters: 98/30″198/8″; 68/10″; 72/20″1×40; 72/2′. Clones of interestexhibiting 111r1+/+, 111r1+/−, and 111r1/— genotypes were passaged onMEFs and culture expanded in preparation for micromass culture. [

Micromass Pre-differentiation Culture. Induced pluripotent stem cellswere subjected to a 15-day, high-density micromass culture to achievedifferentiation toward a mesenchymal state. Cells were cultured inserum-free differentiation medium consisting of high glucose DMEM, NEAA,2-ME, ITS+ (insulin, transferrin, selenium) premix supplement (BD), 25ng/ml gentamicin, 50 μg/m1L-ascorbic acid-phosphate, and 40lag/m1L-proline. On days 3-5 only, medium was supplemented with 100 nMdexamethasone (Sigma) and 50 ng/ml murine BMP-4 (R&D Systems).Micromasses were dissociated on day 15 with pronase and type IIcollagenase in order to attain a single cell suspension, and flowcytometry was used to sort GFP+ cells based on Col2a1 reporterexpression. GFP+ cells were plated in monolayer on gelatinized vesselsand cultured in differentiation medium supplemented with 4 ng/ml bFGF(Roche) and 10% FBS for 2-3 passages. Cells were subsequently utilizedin monolayer assays for functional Illrl protein or in cartilage tissueengineering experiments evaluating the utility of these cells as asource for IL-1-protected tissue regeneration.

Flow Cytometry. Induced pluripotent stem cells and pre-differentiatedcells were trypsinized, washed in PBS, and resuspended in PBS with 1%FBS supplemented with 5 ug/ml anti-mouse CD16/32 (Biolegend) to blocknon-specific immunolabeling. Cells were then immunolabeled with eitheran Armenian hamster anti-mouse CD121a antibody conjugated tophycoerythrin or an isotype control (Biolegend). Cells were washed threetimes then subjected to flow cytometry analysis to determine thepresence or absence of Illrl.

NF-KB Activity Assay. A lentiviral construct containing 5 tandem NF-KBresponse elements(5′-GGAAATTCCCGGAAAGTCCCCGGAAATTCCCGGAAAGTCCCCGGAAATTCCC-3′ (SEQ ID NO:5)) upstream of firefly luciferase was generated by cloning thefollowing sequence upstream of the minimal CMV promoter in pGL3Basic(Promega) and then sub-cloning the cassette into a lentiviral expressionvector. Lentivirus was generated by co-transfecting 4 μg of the clonedtransfer vector, 3 μg of psPAX2 (Addgene 12260) and 1.2 lug of pMD2G(Addgene #12259) into 293T cells cultured at confluence in the well of a6-well plate using Lipofectamine 2000. The next day, medium from 293Tlentivirus producer cells was changed and conditioned medium containinglentivirus was collected 36 and 60 hours after transfection. Thelentiviral supernatant was filtered through 0.45 um cellulose acetatefilters and stored at −80° C. until use.

Pre-differentiated cells were transduced by supplementing culture medium1:1 with viral supernatant as well as 4 ug/ml polybrene and incubatingthe cells in the presence of the virus overnight. Transduced cells wereexpanded, passaged, and then treated with IL-1. At the indicated timepoints, samples were lysed and assayed for luminescence using a BrightGlo luminescence kit according to manufacturer's instructions.Luminescence normalized to background levels of 0 ng/ml ILI treatmentwere used to report induction of NF-KB transcriptional activity.

Chondrogenesis in Aggregate Culture System. Passage 2 pre-differentiatedcells were trypsinized and resuspended in differentiation mediumsupplemented with 100 nM dexamethasone and 10 ng/ml TGF-(33 (R&DSystems) at a density of 1 e6 cells/ml. Aggregate cultures were producedby placing 250,000 cells in each well of a round-bottom 96-well plate.Cells were pelleted and cultured for 27 days prior to inducing aninflammatory assault utilizing an established in vitro osteoarthritismodel (Willard et al., (2014) Arthritis Rheumatol. 66:30623072). At day27, cells were cultured in differentiation medium supplemented with 1ng/ml IL-la and without dexamethasone and TGF-I33. Control aggregatesreceived 0 ng/ml IL-la. Three days later, aggregate cultures and culturesupernatant samples were harvested for gene expression, biochemical, andhistological analyses.

Biochemical Analyses. Samples used for biochemical analyses wereharvested, rinsed with DPBS, and stored at −20° C. until testing.Aggregate culture samples were digested in papain (125 μg/ml; Sigma) at65° C. overnight. Digested samples were then analyzed using thepicogreen assay (Life Technologies) to measure double-stranded DNA, theortho-hydroxyproline assay (Woessner, (1961) Arch. Biochem. Biophys.93:440-447) for measuring total collagen content, and thedimethylmethylene blue assay (Farndale et al., (1986) Biochim. Biophys.Acta 883:173-177) for measuring the total sulfated glycosaminoglycancontent of constructs (n=4-6 per group).

Gene Expression. Samples for gene expression analysis were harvested,rinsed in DPBS, and frozen at −80° C. until further processing. TotalRNA was isolated per manufacturer's recommendations (Norgen Biotek)following tissue homogenization with a pestle. Reverse transcription wasperformed using the superscript VILO cDNA synthesis kit (LifeTechnologies) per manufacturer's instructions. Quantitative RT-PCR wasperformed with n=4 samples per group on a StepOnePlus using Power Sybr(Applied Biosystems, Inc.) per manufacturer's instructions. Fold changeswere determined relative to a reference group cultured without IL-1 aand by using 18s rRNA as a reference gene. Gene expression was probedusing the primer pairs listed in Table 1.

TABLE 1 Primers Pairs used in qRT-PCR gene expression assays. TargetForward Primer SEQ ID NO: r18s 5′-CGGCTACCACATCCAAGGAA-3′ 6 Acan5′-GCATGAGAGAGGCGAATGGA-3′ 7 Adamts4 5′-GACCTTCCGTGAAGAGCAGTGT-3′ 8Adamts5 5′-GCCCACCCAATGGTAAATCTTT-3′ 9 Cc12 5′-GGCTCAGCCAGATGCAGTTAA-3′10 Col2a1 5′-TCCAGATGACTTTCCTCCGTCTA-3′ 11 Elf35′-GGCCCTCATGGCTGCCACCT-3′ 12 IL6 5′-GAGGATACCACTCCCAACAGACC-3′ 13 Mmp95′-CGAACTTCGACACTGACAAGAAGT-3′ 14 Mmp 13 5′-GGGCTCTGAATGGTTATGACATTC-3′15 Target Reverse Primer SEQ ID NO: r18s 5′-GGGCCTCGAAAGAGTCCTGT-3′ 16Acan 5′-CTGATCTCGTAGCGATCTTTCTTCT-3′ 17 Adamts45′-CCTGGCAGGTGAGTTTGCAT-3′ 18 Adamts5 5′-TGACTCCTTTTGCATCAGACTGA-3′ 19Cc12 5′-CCTACTCATTGGGATCATCTTGCT-3′ 20 Col2a15′-AGGTAGGCGATGCTGTTCTTACA-3′ 21 Elf3 5′-TTGGGATCTTGTCTGAGGTCCTGGA-3′ 22IL6 5′-AAGTGCATCATCGTTGTTCATACA-3′ 23 Mmp9 5′-GCACGCTGGAATGATCTAAGC-3′24 Mmp 13 5LAGCGCTCAGTCTCTTCACCTCTT-3′ 25

Histological Processing. Samples for histology were rinsed in DPBS uponharvest, fixed in 4% paraformaldehyde for 24 hours, paraffin embedded,and sectioned at 10 [tm thickness. Samples were stained withSafranin-O/fast green/hematoxylin using standard protocols.

Analyses of Culture Supernatants. Nitric oxide, Prostaglandin E2, sGAG,MMP activities were measured in medium samples (n=4) collected afterIL-la treatment as previously described (McNulty et al., (2011)Connective Tissue Research 52:523-533). As with biochemical samples,sGAG in medium samples was assessed using the DMMB assay. MMP activitywas assessed after activating latent MMPs in supernates with p-APMA.Total specific MMP activity was measured as the difference influorescence arising from cleavage of a quenched fluorogenic substrate(DAB-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys-Flu, Sigma) in samples incubatedwith a broad-spectrum MMP inhibitor GM6001 and a scrambled negativecontrol peptide (EMD Biosciences Inc.). NO and PGE2 were assayed usingcommercially available kits (R&D Systems) following manufacturer'sinstructions.

Statistical Analyses. Statistical analysis was performed in theStatistica 7 software package using ANOVA with Fisher's protected leastsignificance difference post-hoc test with a=0.05. For qRT-PCRcomparisons, fold change values were log-transformed prior tostatistical analysis. Average group values and standard errors of themeans were calculated in the logarithmic space prior to transformingdata to linear values for reporting fold changes.

Example 2 Results—Clonal Isolation and Confirmation of Illrl FunctionalDeficit

Forty-one clones were isolated and screened after single celldeposition. Of these, three were found to possess the Illrl+/− genotypewhile four possessed the Illrl−/− genotype (FIG. 5A). Sanger sequencingof the PCR product from the Illrl−/− clones indicates the expecteddeletion of approximately 790 base pairs, resulting in excision of thesignal peptide for both annotated Illrl isoforms (FIG. 5B). Flowcytometry demonstrated that wild-type cells in the pluripotent state donot express Illrl (FIG. 5C). However, in select clones that werechondrogenically differentiated, a uniform shift occurred in thewild-type (FIG. 5C) population after staining with the anti-Illrlantibody, suggesting low but consistent expression of Illrl on the cellsurface. The Illrl+/− population also displayed a uniform shift, withroughly half the intensity of Illr1+/+ cells, demonstrating reducedexpression of Illrl protein after loss of one functional allele. Cellspossessing the Illrl−/− genotype lacked any positive staining for Illrl(FIG. 5C). The absence of the Illrl receptor on the cell surfaceresulted in a functional deficiency, as indicated by an absence of NF-KBactivity after IL-la stimulation, whereas stimulated wild-type andIllr1+/− cells exhibited a 6.3- and 4.8-fold induction, respectively(FIG. 5D).

Example 3 Cartilage Engineered from CRISPR/Cas9-Edited iPSCs isProtected from IL-La

Gene expression assays demonstrated that IL-la (1 ng/ml) inducedsignificant upregulation of catabolic gene products and markers ofinflammation in aggregates derived from cells with intact Illrl (FIG.6). Cc12 and 116, soluble mediators of OA and sentinel markers ofinflammation, were elevated over 50-fold at 72 hrs in the Illrl+/+ andIllrl+/− aggregates (p<10e-6). Expression of catabolic enzymesresponsible for cartilage matrix degradation, such as Adamts4, Adamts5,Mmp9, and Mmp13, were significantly upregulated in aggregates generatedwith functional Illrl (p<0.007). Expression of Elf3, a transcriptionfactor responsible for cytokine-induced suppression of type II collagen(Peng et al., 2008), was also upregulated in Illrl+/+ and Illrl+/−aggregates (p<10e-6). This corresponded to a concomitant reduction inCol2a1 expression in the same aggregates after IL-la induction(p<10e-6). Furthermore, A can expression was suppressed after IL-latreatment in Illrl+/+ and Illrl+/− aggregates as well (p<10e-6).However, soluble markers of inflammation (p>0.30), catabolic enzymes(p>0.12), and pro-inflammatory transcription factors were notupregulated in Illrl−/− aggregates (p>0.64). Moreover, Col2a1 and Acanexpression were unaffected by IL-la induction (p>0.07, FIG. 6).

In accordance with the observed changes at the transcriptional level,treatment with IL-la resulted in an altered biochemical composition ofcartilage aggregates generated from the Illrl+/+ and Illrl+/− genotypes.Treatment with IL-1 had no effect on DNA content in aggregates (FIG.7A). Interestingly, DNA content in Illrl+/− pellets was significantlyhigher than DNA content in aggregates derived from Illrl+/+ or Illrl−/−cells, possibly due to a higher level of proliferation or cell survivalin the clone chosen for these experiments. Concomitant with thisincreased DNA content, aggregates derived from Illrl+/− displayedincreased accumulation of sGAG and total collagen. Despite thisincreased accumulation, Illrl+/− derived cartilage remained highlyresponsive to IL-la. Sulfated GAG was found to be significantlydependent on IL-la treatment and genotype, with aggregates derived fromIllrl+/+ or Illrl+/− clones losing over 65% of sGAG or sGAG/DNA(p<10e-6, FIG. 7B, FIG. 7D). Aggregates engineered fromCRISPR/Cas9-edited Illrl−/− cells were protected from the IL-latreatment, with no significant difference in sGAG or sGAG/DNA contentassociated with IL-la treatment (p>0.95). No significant differences dueto IL-la were found for total collagen or total collagen/DNA (FIG. 7C,FIG. 7E).

Histological findings support the changes observed in matrix compositionfrom biochemical analyses (FIG. 7F). A GAG-rich matrix developed in allgenotypes after maturation of engineered cartilage. As suggested by thebiochemical data, larger aggregates developed from Illrl+/− cells.However, a marked reduction of Safranin-O staining was observed inaggregates with intact 11 1 rl after IL-1 treatment, consistent with thebiochemical measurement of loss of sGAG for both Illrl+/+ and Illrl+/−derived aggregates.

Culture media collected from Illrl+/+ and Illrl+/− samples displayedfeatures characteristic of a degenerative environment after a 72-hourtreatment with IL-la, whereas media collected from Illrl−/− samplesexhibited no signs of IL-la-responsiveness (FIGS. 8A-8D). Specific MMPactivity was significantly elevated in media collected fromIL-la-treated Illrl+/+ and Illrl+/− aggregates (FIG. 8A), likelycontributing to the elevated levels of sGAG detected in the same mediasamples (FIG. 8B). The accumulation of significantly higher levels ofsGAG in these samples is consistent with the observed loss of sGAG inengineered cartilage derived from Il 1 rl+/+ and Il 1 rl+/− cells.Furthermore, the higher levels of PGE2 and total nitric oxide speciesfound in 111r1+/+ and Illrl+/− samples (FIG. 8C and FIG. 8D) reflectsthe inflammatory state IL-la was capable of inducing whenIllrl-competent cells were used to engineer cartilage, while IL-la didnot affect the of the Illrl−/−-derived cartilage.

This work demonstrates the utility of programmable nucleases forapplications in tissue engineering and regenerative medicine bydeveloping stem cells with customized properties at the scale of thegenome. Using targeted gene editing nucleases, pluripotent stem cellswere engineered with the trait of IL-1-resistance by deleting the Illrlsignal peptide sequence. CRISPR/Cas9-mediated editing of the Illrl locusresulted in complete loss of IL-1 signaling by all measures evaluated.Cartilage derived from CRISPR/Cas9-edited pluripotent stem cellsdisplayed the capacity to withstand treatment with 1 ng/ml IL-1. Takentogether, these results indicate that genome editing serves as aneffective means for generating stem cells with application-specificfeatures pertinent to tissue regeneration, maintenance, and repair. Thepro-inflammatory environment generates a disruption of the collagenousnetwork that normally resists swelling of the tissue and also results incatabolism of the intertwined proteoglycan mesh, leading to severecartilage erosion.

Cartilage derived from either 111r1+/+ or Illrl+/− cells was highlysusceptible to this in vitro model of OA, as shown by extreme loss ofsGAG and production of degradative enzymes such as MMPs as well aspro-inflammatory mediators including PGE2 and NO. Furthermore, cartilagederived from 111r+/+ and 111r1−/− cells showed a significant inductionof a pro-inflammatory gene transcriptional program after 1L-1 treatment.However, cartilage derived from CRISPR/Cas9-engineered cells lackingfunctional Illrl demonstrated complete protection from IL-1 treatment,including preservation of extracellular matrix constituents as observedhistologically and biochemically, as well as a lack of induction ofcatabolic enzymes and pro-inflammatory mediators.

The results show that genome editing with CRISPR/Cas9 yielded thedesired genomic modification in more than 10% of isolated clones in anunselected population. This method allows for site-specific genedeletion independent of targeting donor vectors that harbor gene trapsor loxP sites that facilitate subsequent Cre-mediated excision. Thus,this approach overcomes the need for ectopic overexpression ofselectable markers or Cre recombinase, and allows for direct, efficientgene editing.

The data suggests that the presence of IL-1Ra is sufficient to combatpathologic levels of IL-1, which may prove challenging if engineeredtissues remain susceptible to high levels of IL-1 signaling. Cartilageengineered from cells rendered incapable of transducing the IL-1 signalmay serve as an ideal cell-based drug delivery platform, which mayremain intact in the presence of high levels of IL-1 while functioningto protect the surrounding, IL1R1-competent cells from IL-1 signaling bysecreting IL-1Ra into the inflamed microenvironment.

Example 4 Methods—Cc12

A treatment of chronic inflammatory diseases such as arthritis wasdeveloped with stem cells that can autonomously execute real-time,programmed responses to pro-inflammatory cytokines. Genome editingnucleases based on the CRISPR/Cas9 platform were used to introducespecific modifications to chromosomal gene sequences in iPSCs. Targetedaddition of transgenes encoding cytokine antagonists was performed tocreate a closed-loop gene circuit based on the inflammation-induciblechemokine Cc12 endogenous locus. Such targeted gene modificationimparted self-regulated, feedback-controlled production of biologictherapy induced by the inflammatory transcriptional program controlledby cytokines such as IL-1 and TNF-a. Repurposing of degradativesignaling pathways toward transient production of cytokine antagonistsenabled engineered cartilage tissue to withstand the action ofinflammatory cytokines and serve as a cell-based autoregulated drugdelivery system. Treatment of genetically engineered iPSCs with eitherIL-la or TNF-a resulted in upregulated transgene transcription inresponse to endogenous Cc12 activation in a dose- and time-dependentmanner. Expression profiles demonstrated rapid induction and subsequentdecay of transgene expression concomitant with attenuation of cytokinesignaling. Cartilage derived from cells autonomously expressinganti-cytokine biologics was protected from cytokine-mediated degradationas compared to cartilage engineered from control cells. This resulted inpreservation of the cartilage ECM as measured by quantitativebiochemical assays and histology. This work demonstrates the utility ofgenome engineering for the development of stem cells with propertiescustomized for cell-based regenerative medicine strategies for thetreatment of chronic inflammatory diseases

Induced pluripotent stem cell derivation and culture. Murine inducedpluripotent stem cells were derived and cultured as previously described(Diekman et al., (2012) Proc. Natl. Acad. Sci. 109:19172-19177).Briefly, tail fibroblasts from adult C57BL/6 mice were transduced with alentiviral vector driving doxycycline-inducible expression of Oct4(Pou5fl), Sox2, Klf4, and c-myc (Carey et al., (2009) Proc. Natl. Acad.Sci. 106:157-162). Pluripotent cells were maintained on mitomycinC-treated mouse embryonic feeders (MEFs; Millipore) in medium comprisedof high glucose Dulbecco's modified Eagle's medium (DMEM) supplementedwith L-glutamine, sodium pyruvate, 20% fetal bovine serum, 100 nMminimum essential medium non-essential amino acids (NEAA; Gibco), 55 1 .. . LM13-mercaptoethanol (2-ME; Gibco), and 1,000 units of leukemiainhibitory factor (LIF; Millipore). A Col2al-GFP reporter construct(Grant et al., 2000) was transfected into cells by Nucleofection, and aclone stably expressing the reporter upon chondrogenic induction wasisolated after G418 selection.

Genome editing and clonal isolation. A plasmid encoding human codonoptimized Streptococcus pyogenes Cas9 (hCas9) was obtained as a giftfrom George Church (Mali et al., 2013) (Addgene plasmid #41815). Totarget hCas9 to the Ccl2 locus, a protospacer targeting the start codonof the Cc12 coding sequence was generated using the followingcomplementary oligonucleotides: sgMcp1-4 S: 5′-cacc GCTCTTCCTCCACCACCATGC-3′ (SEQ ID NO: 26) and sgMcp1-4 AS: 5′-aaacGCATGGTGGTGGAGGAAGAG C-3′ (SEQ ID NO: 27), where lower case bases wereused to clone into BbsI-generated overhangs in the expression vector,and the guanine upstream of the protospacer was included to promoteefficient transcription from the U6 promoter in the expression vector.To produce a single chimeric guide RNA (sgRNA) expression vector,complementary oligonucleotides containing the protospacer sequence werehybridized, phosphorylated, and cloned into an expression vector(Perez-Pinera et al., 2013a) (Addgene plasmid #47108) employing an humanU6 promoter to drive expression of a chimeric Streptococcus pyogenescrRNAAracrRNA sequence containing the aforementioned protospacer. ThegRNA sequence was sgRNA Cc12-4: G CTCTTCCTCCACCACCATGC (SEQ ID NO: 45).

Targeting vectors were produced in which coding sequences for eachtransgene (lucifcrasc, sTNFR1-IgG, or Illra) were cloned directly inplace of the start codon of Cc12. The left homology arm was generated byPCR amplifying the region flanked by the following oligonucleotides frommurine genomic DNA isolated using the DNeasy Blood & Tissue Kit(Qiagen): 5′-AAATTTCTTCTGCACCATGAG-3′ (SEQ ID NO: 28) and5′-CATGGTGGTGGAGGAAGAGAGAGC-3′ (SEQ ID NO:29). The right homology armwas similarly generated and defined by the following oligonucleotides:5′-CAGGTCCCTGTCATGCTTCTG-3′ (SEQ ID NO: 30) and5′-ATCTGGGATGTGATCTTTGACA-3′ (SEQ ID NO: 31). Targeting vectors wereproduced by isothermal assembly using pGL3-Basic (Promega) as abackbone. First, PCR fragments including transgenes followed by a Simianvirus 40 polyadenylation signal sequence (SV40 polyA) were ligated tothe left and right homology arms. Template for the Luciferase transgenewas pGL3-Basic. The template for the sTNFR1-IgG transgene was a vectoras previously described (Bloquel et al., (2004) Human Gene Therapy15:189-201). The template for Illra was cDNA from murine C57B1/6 mice.The resulting vectors were sequence confirmed, then an expressioncassette comprised of the CMV promoter, the hygromycin Bphosphotransferase coding sequence, and a bovine growth hormone polyAwas cloned into each vector between the 3′ end of the SV40 polyAfollowing the transgene and the 5′ end of the right homology arm.

Prior to transfection, iPSCs were trypsinized and subjected to a30-minute feeder subtraction. Lipofectamine 2000 (Life Technologies) wasused following manufacturer's instructions to co-transfect 800 ng ofeach sgRNA and 800 ng hCas9 along with 1.5 [ig of the appropriatetargeting vector into iPSCs freshly plated on MEFs in complete,antibiotic-free iPSC medium in a 6-well plate. The following day, cellswere subjected to selection using 100 μg/ml of hygromycin B (LifeTechnologies). Transfected cells were subcultured on MEFs for 2 weeksprior to clonal isolation.

Clones were isolated either by iterative mechanical picking or bysingle-cell deposition. Single cell deposition was performed using aFACSVantage sorter (Becton Dickson). In preparation for single celldeposition, iPSCs were feeder subtracted prior to culture on 0.1%gelatin for 2 days. Cells were then trypsinized and subjected to a finalfeeder subtraction and then suspended in calcium- and magnesium-freePBS, 1 mM EDTA, 25 mM HEPES, and 1% FBS. Individual cells were thendeposited into MEF-containing wells of a 96-well plate. Clones weresub-cultured on MEFs throughout the screening process. Targetedintegration was assayed by performing junction PCR using theoligonucleotides listed in Table 1 for each target. For the PCR, asubset of each clone was lysed using QuickExtract (Epicentre) accordingto the manufacturer's instructions. The cell lysate was then diluted8-10 fold prior to use as template in a PCR using Q5 polymerase (NEB)according to manufacturer's instruction with the following cyclingparameters: 98/30″198/8″; 68/10″; 72/20″1×35; 72/2′. Clones exhibitingunique and specific product from the junction PCR were propagated onMEFs and until further differentiation. Further analysis of targeting ofthe Cc12 alleles in these clones were performed using the followingoligonucleotide pair: Sury MCP1 FI: 5′-tcccaggagtggctagaaaa-3′ (SEQ IDNO: 32); Sury MCP1 R1: 5′-ccacgacaattcaaaaatgg-3′ (SEQ ID NO: 33).

TABLE 2  Primer pairs used in junction PCR todetermine the presence of targeted integration events on Cc12 alleles.SEQ ID Target Forward Primer NO: IL Ira 5′-TCAGCTGCCTGATCTGAGAA-3′ 34Firefly 5′-TCAGCTGCCTGATCTGAGAA-3′ 34 Luciferase sTNFR1-IgG5′-TCAGCTGCCTGATCTGAGAA-3′ 34 SEQ ID Target Reverse Primer NO: IL Ira5′-AGGTCAATAGGCACCATGTCTA-3′ 35 Firefly 5′-CAGCGTAAGTGATGTCCACCT-3′ 36Luciferase sTNFR1-IgG 5′-CACTCCCTGCAGTCCGTATC-3′ 37

Micromass pre-differentiation culture. Induced pluripotent stem cellswere subjected to a 15-day, high-density micromass culture to achievedifferentiation toward a mesenchymal state. Cells were cultured inserum-free differentiation medium consisting of high glucose DMEM, NEAA,2-ME, ITS+(insulin, transferrin, selenium) premix supplement (BD), 25ng/ml gentamicin, 50 μg/m1L-ascorbic acid-phosphate, and 40μg/m1L-proline. On days 3-5 only, medium was supplemented with 100 nMdexamethasone (Sigma) and 50 ng/ml murine BMP-4 (R&D Systems).Micromasses were dissociated on day 15 with pronase and type IIcollagenase in order to attain a single cell suspension. Dissociatedcells were plated in monolayer on gelatinized vessels and cultured indifferentiation medium supplemented with 4 ng/ml bFGF (Roche) and 10%FBS. Cells were subsequently utilized in monolayer to probe thedynamical response of engineered cells to IL-1 or TNF treatment.Additionally, cells were used to derive engineered cartilage in order toevaluate the utility of these cells as a source forinflammation-protected tissue regeneration.

NF-KB activity assay. A lentiviral construct containing 4 putative NF-KBresponse elements upstream of firefly luciferase was generated bycloning the following sequence:5′-CGGGAAATTCCGCTAGCACTAGTGGGACTTTCCCACTAGTGGGAAATTAGCCCGGGACTTTCCCGTCTCCTCGAGGGGACTTCCCA-3′ (SEQ ID NO: 40) upstream of theminimal CMV promoter in pGL3Basic (Promega) and then sub-cloning thecassette including the luciferase transgene into a lentiviral expressionvector. Additionally, an NF-KB negative regulatory element(NRE—5′-AATTCCTCTGA-3′ (SEQ ID NO: 41)) (Nourbakhsh et al., (1993) EMBOJ 12:451-459) was cloned upstream of the response elements in order toreduce background signal from the luciferase vector. Lentivirus wasgenerated by co-transfecting 2 μg of the cloned transfer vector, 1.5 lugof psPAX2 (Addgene 12260) and 0.6 lug of pMD2G (Addgene 12259) into 293Tcells cultured at confluence in the well of a 6-well plate usingLipofectamine 2000. The next day, medium from 293T lentivirus producercells was changed and conditioned medium containing lentivirus wascollected approximately 36 and 60 hours after transfection. Thelentiviral supernatant was filtered through 0.45 um cellulose acetatefilters and stored at −80° C. until use.

Pre-differentiated cells were transduced by supplementing culture medium1:1 with viral supernatant as well as 41.1 g/ml polybrene and incubatingthe cells in the presence of the virus overnight. Transduced cells wereexpanded, passaged, and then treated with IL-1. At the indicated timepoints, samples were lysed and assayed for luminescence using a BrightGlo (Promega) luminescence kit according to manufacturer's instructions.Luminescence normalized to background levels of no cytokine treatmentwas used to report induction of NF-KB transcriptional activity.

Chondrogenesis in aggregate culture system. Multiple chondrogenesisexperiments were performed using slightly varying experimentalconditions. Each experiment comprised of matched controls cultured underthe same conditions (e.g., passage number, starting cell number persample, length of cartilage maturation period). Passage 2-3pre-differentiated cells were trypsinized and resuspended indifferentiation medium supplemented with 100 nM dexamethasone and 10ng/ml TGF-I33 (R&D Systems) at a density of 1e6 cells/ml. Aggregatecultures were produced by placing cells in wells a u-bottom 96-wellplate (125,000-250,000 cells per well, depending on the experiment) orin 15 ml conical tubes (500,000 cells per tube). Cells were pelleted bycentrifugation at 200×g and cultured for three to four weeks prior totreatment with cytokine (0, 0.1-1 ng/ml IL-la, or 20 ng/ml TNF-a) in theabsence of dexamethasone and TGF-133. Three days later, aggregatecultures and culture supernatant samples were harvested for geneexpression, biochemical, and histological analyses.

Biochemical analyses of engineered cartilage. Samples used forbiochemical analyses were harvested, rinsed with DPBS, and stored at−20° C. until testing. Aggregate culture samples were digested in papain(125 iug/m1; Sigma) at 65° C. overnight. Digested samples were thenanalyzed using the picogreen assay (Life Technologies) to measuredouble-stranded DNA, the ortho-hydroxyproline assay (Woessner, (1961)Arch. Biochem. Biophys. 93:440-447) for measuring total collagencontent, and the dimethylmethylene blue assay (Farndale et al., (1986)Biochim. Biophys. Acta 883:173-177) for measuring the total sulfatedglycosaminoglycan content of tissues (n=3-6 per group).

Gene expression. Samples for gene expression analysis were rinsed inDPBS, lysed in cell lysis reagent (Norgen Biotek) and frozen at −80° C.until further processing. Total RNA was isolated per manufacturer'srecommendations (Norgen Biotek). Engineered cartilage samples were firsthomogenized with a pestle. Reverse transcription was performed using thesuperscript VILO cDNA synthesis kit (Life Technologies) permanufacturer's instructions. Quantitative RT-PCR was performed withn=3-4 samples per group on a StepOnePlus using Power Sybr (AppliedBiosystems, Inc.) per manufacturer's instructions. Fold changes weredetermined relative to a reference group cultured without IL-la and byusing 18s rRNA as a reference gene. Gene expression was probed using theprimer pairs listed in Table 3.

TABLE 3  Primer pairs used in qRT-PCR gene expression assays SEQ IDTarget Forward Primer NO: r18s 5′-CGGCTACCACATCCAAGGAA-3′ 6 Acan5′-GCATGAGAGAGGCGAATGGA-3′ 7 Adamts4 5′-GACCTTCCGTGAAGAGCAGTGT-3′ 8Adamts5 5′-GCCCACCCAATGGTAAATCTTT-3′ 9 Cc12 5′-GGCTCAGCCAGATGCAGTTAA-3′10 Co12a1 5′-TCCAGATGACTTTCCTCCGTCTA-3′ 11 ILIm5′-GTCCAGGATGGTTCCTCTGC-3′ 40 IL6 5′-GAGGATACCACTCCCAACAGACC-3′ 13 Mmp95′-CGAACTTCGACACTGACAAGAAGT-3′ 14 Mmp13 5′-GGGCTCTGAATGGTTATGACATTC-3′15 sTNFR1 5′-ATTGGACTGGTCCCTCACCT-3′ 41 SEQ ID Target Reverse Primer NO:r18s 5′-GGGCCTCGAAAGAGTCCTGT-3′ 16 Acan 5′-CTGATCTCGTAGCGATCTTTCTTCT-3′17 Adamts4 5′-CCTGGCAGGTGAGTTTGCAT-3′ 18 Adamts55′-TGACTCCTTTTGCATCAGACTGA-3′ 19 Cc12 5′-CCTACTCATTGGGATCATCTTGCT-3′ 20Co12a1 5′-AGGTAGGCGATGCTGTTCTTACA-3′ 21 IL Irn5′-TCTTCCGGTGTGTTGGTGAG-3′ 42 IL6 5′-AAGTGCATCATCGTTGTTCATACA-3′ 23 Mmp95′-GCACGCTGGAATGATCTAAGC-3′ 24 Mmp13 5′-AGCGCTCAGTCTCTTCACCTCTT-3′ 25sTNFR1 5′-CACTCCCTGCAGTCCGTATC-3′ 37

Enzyme-Linked Immunosorbent Assays. Media samples used in ELISAs werecollected from wells and stored at −20° C. or −80° C. until used.Reagents for ELISAs were purchased from R&D and used according tomanufacturer's recommendations.

Histological processing. Samples for histology were rinsed in DPBS uponharvest, fixed in 4% paraformaldehyde for 24 hours, paraffin embedded,and sectioned at 10 pm thickness. Samples were stained withSafranin-O/fast green/hematoxylin using standard protocols.

Statistical analysis. Statistical analysis was performed in theStatistica 7 software package using ANOVA with Fisher's protected leastsignificance difference post-hoc test with a=0.05. For qRT-PCRcomparisons, fold change values were log-transformed prior tostatistical analysis. Average group values and standard errors of themeans were calculated in the logarithmic space prior to transformingdata to linear values for reporting fold changes.

Example 5 Clonal Isolation and Screening

The goal was to reprogram iPSCs with the capacity to respond to aninflammatory transcription stimulant with potent and autonomouslyregulated anti-cytokine production (FIG. 9). As such, transgenesencoding a firefly luciferase transcriptional reporter or a cytokineantagonist, either Illra or sTNFR1-IgG, were targeted to the Cc12 locususing the CRISPR/Cas9 gene editing platform. After hygromycin selectionand junction PCR screening, multiple clones were identified to possesstargeted integration events at the Cc12 locus. For the luciferasetransgene, 2 out of 11 clones initially screened after mechanicalisolation displayed evidence of targeted integration. Afterwards, one ofthese clones was subcloned, and 6 out of 10 subclones displayed clearevidence of targeted integration (2 of these are displayed in FIG. 10).For Illra, 4 out of 34 clones were positive and for sTNFR1-IgG, 3 out of44 possessed the targeted integration event (FIG. 10). Subsequent PCRanalyses were performed to probe whether one or both Cc12 alleles weretargeted in these clones of interest. In all cases, only one allelecontained the targeted integration event. Furthermore, Sanger sequencingwas performed on PCR products to determine whether the remaining Cc12allele lacking targeted integration was disrupted by CRISPR/Cas9 at thestart codon. At least one clone for each transgene group (luciferase,Illra, and sTNFR-IgG) was identified with an intact Cc12 start codon.

Example 6 Characterization of Responsiveness

Clones of interest (referred to as Cc12-Luc, Cc12-Illra, or Cc12-sTNFR1lines as appropriate for each integrated transgene) were cultureexpanded on MEFs and then pre-differentiated in micromass culture.First, whether targeted transgene at the Ccl2 start codon would enablecytokine-inducible transgene expression was evaluated. As a point ofreference, wild-type (WT) cells were treated with a range of TNF-aconcentrations (0.2-20 ng/ml), and mRNA samples were collected at 4, 12,24, and 72 hours (FIG. 11A). Cc12 gene expression was evaluated byqRT-PCR. At all TNF-a concentrations tested, Ccl2 gene expression waselevated at each time point. In the 2 and 20 ng/ml groups, Cc12 geneexpression continued to increase through the 72 hour TNF-a treatmentwindow.

Next, using two Cc12-luciferase cell lines, luciferase expression wasinduced by stimulating cells with 20 ng/ml TNF-a to evaluate whethertransgene expression reflected endogenous Cc12 expression in WT cells.Relative luminescence measurements indicated that transgene expressionin both clones was indeed stimulated by cytokine and increased acrossthe 72-hour TNF-a treatment period (FIG. 11B), consistent with findingsfrom TNF-induced Cc12 expression in WT cells.

The responsiveness of the engineered cells endowed with Cc12-drivenanti-cytokine transgenes as probed. The experiments were performed byevaluating gene expression and transgene production in the Cc12-sTNFR1group, as lack of this human transcript and protein in the murine cellpopulations would allow for direct conclusions regarding production fromthe Ccl2 locus, whereas murine Illra can be produced from its ownendogenous gene as well as the Cc12 locus in the engineered cells.

Initially, a time course and dose response experiment were performed, inwhich Cc12-sTNFR1 and wild-type cells were treated with a range of TNF-aconcentrations (0.2-20 ng/ml) for a variety of times (4, 12, 24, and 72hours). The responsiveness of the engineered cells was evaluated bymeasuring the expression of the sTNFR1 transgene at both the mRNA andprotein levels by qRT-PCR and ELISA, respectively. Furthermore, theexpression of 116 at the mRNA level by qRT-PCR was measured tocharacterize the state of inflammation the WT and engineered Ccl2-sTNFR1cells experienced.

As early as 4 hours after TNF-a treatment, the 2 and 20 ng/ml treatmentsresulted in significant upregulation of 116 transcription in both the WTand Cc12-sTNFR1 cells, while 0.2 ng/ml did not render significantupregulation (FIG. 12A). At the 12 hour time point, 116 expression wassignificantly elevated at all TNF-a concentrations in WT cells; however,116 was only significantly upregulated in the Cc12-sTNFRI engineeredcells at the 20 ng/ml level of TNF-a treatment (FIG. 12B). Even at the20 ng/ml level of treatment, engineered cells showed a significantlylower level of 116 induction than WT cells. At the 24 hour time point,the medium and high concentrations of TNF-a drove an upregulation of 116in WT cells, but only the high 20 ng/ml concentration resulted insignificant upregulation of 116 in the sTNFR1 engineered cells (FIG.12C). By the 72 hour time point, all three doses of TNF-a resulted insignificant upregulation of 116 in the WT cells, while TNF-a treatmentonly induced an upregulation of 116 in the Ccl2-sTNFRI cells at the 20ng/ml treatment level (FIG. 12D).

To evaluate whether the observations of 116 gene expression reflect thegeneral state of inflammation in these cells, WT and Cc12-sTNFR1 cellswere transduced with a lentiviral vector delivering an NF-KBluminescence reporter. These cells were treated with 0 or 20 ng/ml TNF-aand after 24, 48, and 72 hours, lysed cells to measure luminescence asan output for NF-xl3 transcriptional activity. At the 24 hour timepoint, the NF-KB transcriptional activity was upregulated in both WT andCc12-sTNFR1 cells. However, at the 48 and 72 hour time points, a sharpdecline in NF-KB transcriptional activity was observed in engineeredcells expressing sTNFR1 under control of the Cc12 locus (FIG. 13A).Taken together with the 116 mRNA qRT-PCR results, these data indicatethat the Cc12-sTNFR1 cells are capable of attenuating the TNF-a-inducedregulation of 116 as well as a more general inflammatory state.Furthermore, these results suggest that, after three days of TNF-atreatment, the cells are capable of antagonizing even a high (20 ng/ml)concentration of TNF-a, while control WT cells remain in a state ofinflammation even after treatment with only 0.2 ng/ml TNF-a.

To ascertain whether this attenuation could be mediated bycytokine-inducible production of the TNF-a antagonist sTNFR1 from theengineered cells, the expression of sTNFR1 transgene was measured inparallel with 116 expression. sTNFR1 expression was rapidly upregulatedat the 4 hour time point (FIG. 13B). In the groups treated with 0.2 and2 ng/ml of TNF-a, transgene expression began to decline between the 4and 12 hour time points, in accordance with the decreased 116 expression(FIG. 12A, FIG. 12B). This transition likely reflects an attenuatedstate of inflammation after low and medium treatment of TNF-a.Cc12-driven sTNFR1 expression continued to increase through the 24 hourtime point at the high TNF-a treatment, but this level declined rapidlytoward baseline values at the 72 hour time point, consistent with thepersistent state of inflammation at 24 hours that largely resolved by 72hours as suggested by the 116 and NF-KB transcription data FIG. 12C,FIG. 12D and FIG. 13A). In accordance with these qRT-PCR data, increasedaccumulation of sTNFR1 was measured in culture media over time in adose-dependent fashion (FIG. 13C).

From this, even a low concentration of TNF-a is capable of inducingsTNFR1 expression, indicating that low in vitro doses of TNF-a caninduce transgene expression in the engineered cell population. Thissuggests that cells remain attuned to low levels of TNF-a in themicroenvironment, indicating that basal expression of sTNFR1 does notabolish the cell's ability to detect low concentrations of the cytokine.These data also demonstrate that the engineered cells are capable ofmodulating therapeutic output of sTNFR1 in a cytokine dose-dependentmanner, spanning a dynamic range across at least three orders ofmagnitude. Furthermore, the anti-cytokine therapy appears to beauto-regulated, as expression of sTNFR1 mRNA declined as theinflammatory state of the engineered cells resolved in response tosTNFR1 production.

As a follow-up to these analyses, iterative stimulation of Cc12-drivensTNFR1 and Illra cells in monolayer were performed with either 0 ng/mlcytokine, 0.1 ng/ml IL-la or 20 ng/ml TNF-a. After 24 hours, thecytokine-containing medium was exchanged for cytokine-free medium, andsamples were collected. Three days later, cells were stimulated withcytokine again to establish the capacity of the cells to respond torecurrent stimulation with cytokine. sTNFR1-engineered cells displayed abasal level of production of less than 3 ng/ml (FIG. 14A, FIG. 14B).Engineered cells displayed the capacity to rapidly secrete sTNFR1 aftereither IL-la or TNF-a stimulation (FIG. 14A).

Withdrawal of cytokine-containing medium resulted in a rapid decline insTNFR1 accumulation over subsequent collection periods, irrespective ofwhether IL-la or TNF-a served as the stimulant. In both cases,production of sTNFR1 decreased to basal levels within 48 hours ofremoving cytokines (FIG. 14A).

When treated with 0.1 ng/ml IL-la, which sTNFR1 would not be expected toantagonize, there was approximately 300-fold stimulation of sTNFR1production (FIG. 14B) to −630 ng/ml. When treated with 20 ng/ml TNF-a,production of sTNFR1 increased only approximately 50-fold over basallevels to −90 ng/ml. In a similar vein, treatment of Cc12-Il1ra cellswith IL-la resulted in an increase of Illra protein in the medium ofapproximately 30-fold over basal levels of expression to −180 ng/ml,whereas treatment with TNF-a resulted in an increase of approximately88-fold to −570 ng/ml (FIG. 14C). Thus, in the case of both Illra- andsTNFR1-expressing cells, either IL-la or TNF-a are capable of potentlyinducing transgene expression. However, a lower fold induction isachieved when an effective, antagonizing therapy is produced in responseto the stimulatory cytokine. The expression of transgenes may beauto-regulated in response to attenuation of the inducing signal. Cellscan continue to respond to cytokines by producing additional therapyafter previous exposure in a robust manner at the 24 hour timescale.

Example 7 Auto-Regulated Production of Cytokine Antagonists ProtectsEngineered Cartilage from IL-1- and TNF-Mediated Catabolism

After establishing that the engineered cells indeed express transgenesin a cytokine-inducible manner and that Ccl2-driven sTNFR1 mounts atunable and effective response against even a high dose of TNF-a inmonolayer experiments, whether tissues engineered from the genomeedited, designer stem cells could overcome the degenerative effects ofTNF-a, and IL-la was investigated. Toward this end, pre-differentiatedcells from WT, Cc12-Luc, Cc12-Illra, and Cc12-sTNFR1 clones were furtherdifferentiated toward the chondrocyte lineage for the production ofengineered cartilage tissue. Engineered tissues from WT and Cc12-Luccell lines were treated with 0 ng/ml cytokine, 0.1-1 ng/ml IL-la or 20ng/ml TNF-a. Engineered tissues from Cc12-Il1ra and Cc12-sTNFR1 celllines were treated with only IL-la or TNF-a, respectively, at the sameconcentrations as WT and Cc12-Luc tissues.

The cartilage derived from WT and Luc cell lines was severelydeteriorated in response to this 72 hour cytokine treatment aftercartilage maturation. The changes in gene expression induced by 1 ng/mlIL-la or 20 ng/ml TNF-a were measured by qRT-PCR (FIG. 15 and FIG. 16,respectively) and observed significant upregulation of a variety ofmarkers of inflammation, such as Cc12 and 116, as well as degradativeenzymes, such as matrix metalloproteinases and aggrecanases.Furthermore, significant suppression of collagen type 2 al (Col2a1) andaggrecan (Acan), is noted in cartilage engineered from either WT orCc12-Luc cells. Biochemically cartilage derived from these control celllines reflected a loss of sulfated glycosaminoglycan (sGAG), a majorcomponent of articular cartilage critical to proper tissue function, inresponse to both concentrations of IL-la and to 20 ng/ml TNF-a (FIG.17A, FIG. 17B, FIG. 17C, and FIG. 17D).

Cartilage derived from Cc12-Illra or Cc12-sTNFR1 cells displayed amarkedly different response to cytokine treatment at the gene expressionlevel. Tissue generated from both the Cc12-Illra and Cc12-sTNFR1 celllines demonstrated lower induction levels of inflammatory anddegradative gene products as compared to cartilage engineered from WT orCc12-Luc cell lines (FIG. 15 and FIG. 16, respectively). In some cases,these genes were still significantly upregulated relative to tissuestreated with 0 ng/ml cytokine. In the case of Cc12-sTNFR1, cartilageaggregates displayed resilience after the 72 hours of treatment withTNF-a, with no suppression of collagen type 2 al or aggrecan. Thepreservation of a more homeostatic gene expression profile was found tobe consistent with the biochemical composition of cartilage aggregatesengineered from the Cc12-sTNFR1 cell line, which demonstratedpreservation of sGAG in the tissue even after treatment with 20 ng/mlTNF-a, as reflected from biochemical and histologic data (FIG. 17A, FIG.18). TNF-a was shown to have induced secretion of sTNFR1, as samplestreated with 20 ng/ml TNF-a produced 18.45±0.17 ng/ml sTNFR1 and thosecultured in the absence of TNF-a produced only 3.31±0.17 ng/ml sTNFR1.

However, Cc12-driven expression of Il 1 ra was not sufficient to protectagainst the suppression of the extracellular matrix constituents Col2a1and Acan by 1 ng/ml IL-la, and this, coupled with the increasedexpression of degradative enzymes, resulted in loss of a significantfraction of sGAG in the engineered tissue (FIG. 17B, FIG. 17C, and FIG.17D). At the 0.1 ng/ml IL-la level, cartilage derived from engineeredCc12-Illra cells were less susceptible to degradation than tissuederived from control Cc12-Luc cells, though sGAG loss normalized tototal DNA content was still statistically significant after cytokinetreatment in both tissue types (FIG. 17). This protection was impartedby the cytokine induced expression of 20.50±0.67 ng/ml Illra, which washigher than the basal expression of 1.82±0.24 ng/ml observed in theengineered cells or 0.88±0.25 ng/ml observed in Ccl2-Luc cells. Theeffect of 0.1 ng/ml IL-la on cartilage derived from WT cells was nottested.

Treatment with a range of TNF concentrations spanning three orders ofmagnitude resulted in differential induction of transgene transcription.The concomitant decay in transgene expression and transcription ofmarkers of inflammation such as 11-6 suggests that cells were capable ofautonomously tuning expression of the transgene. The AU-rich elements inthe 3′-UTR were uncoupled by insertion of the transgene cassette, whichincluded a drug-selectable marker as well as polyadenylation sequences.Expression of the transgenes did decay after resolution of cytokinestimulation, which came about by transgene therapy or simple withdrawalof cytokine. In some embodiments, reservation of the AU-rich elements inthe transgene cassette may provide a means whereby even more rapiddeclines in transgene expression may be achieved.

Example 8 ADAMTS-5

Customized iPSCs are generated where the chromosomal loci of catabolicgenes that are activated by TNF via NF-KB are edited. In particular,functional deficiencies of the enzyme Adamts5 prevent cartilagedegeneration in mouse models of osteoarthritis, and ADAMTS-5 inhibitorsare a primary area of pharmaceutical research for arthritis therapies.Synthetic gene-editing nucleases are custom-designed to edit thechromosomal locus of Adamts5 by inducing a targeted double-strand breakat a specific site within that locus. The chromosomal locus of Adamts5is disrupted, and a synthetic expression cassette driving TNF-inducibletranscription of sTNFR1 is inserted into the locus (see FIG. 2). ThesTNFR1 expression cassette is integrated into the iPSC chromosomal DNAvia either lentiviral transduction or targeted genome editing of theAdamts5 locus. Genome engineering with different gene targeting vectorsprovided as templates for homology-directed repair facilitate theintegration of the synthetic expression cassette into the Adamts5 locus.

ADAMTS-5-deficient iPSCs are engineered to possess the NF-xl3 responsivetranscriptional control system driving expression of either luciferaseor sTNFR1 from the Adamts5 locus. iPSC clones are screened via genomicPCR and Southern Blot, and successful targeting is confirmed viasequencing of the edited locus. After stimulation with variousconcentrations and durations of TNF, luciferase reporter assayscharacterize the real-time responsiveness of the system. sTNFR1 gene andprotein expression are measured via qRT-PCR and ELISA. To assess theability of the cells to modulate inflammation, gene and protein levelsof NF-KB targets, such as pro-inflammatory cytokines, MMPs, and ADAMTSfamily members, are analyzed in parallel. These methods are applied tohuman iPSCs or adult stem cells.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

1. A composition for treating a subject having or suspected of having adisease, the composition comprising a modified cell comprising amodified endogenous gene, wherein an endogenous gene or fragment thereofis replaced with a transgene using a CRISPR/Cas9 system to generate themodified endogenous gene, the modified cell having an altered responseto a cell signal or stimulus.
 2. The composition of claim 1, wherein thealtered response to the cell signal or stimulus comprises an activationof the transgene.
 3. The composition of claim 1 or 2, wherein thetransgene encodes a transcription factor or a therapeutic molecule. 4.The composition of claim 3, wherein the transgene encodes atranscription factor and the activation of the transcription factoractivates or downregulates a signaling pathway in response to the cellsignal or stimulus as compared to the response to the cell signal orstimulus by the unmodified endogenous gene.
 5. The composition of claim4, wherein the activation of the transcription factor activates ananti-inflammatory response and the unmodified endogenous gene activatesan inflammatory response.
 6. The composition of claim 5, wherein thecoding region of the endogenous gene is replaced with the coding regionof a transgene and the coding region of the transgene is operably linkedto the promoter of the endogenous gene.
 7. The composition of claim 6,wherein the modified cell comprises self-regulated feedback control ofanti-cytokine therapy to a subject.
 8. The composition of claim 1,wherein the endogenous gene is Cc12 or ADAMTS-5.
 9. The composition ofany one of claims 1 to 8, wherein the transgene is sTNFR1 or IL-1Ra. 10.The composition of claim 3, wherein the transgene encodes a therapeuticmolecule.
 11. The composition of claim 10, wherein the modified cellproduces therapeutic molecules in response to the cell signal orstimulus.
 12. The composition of claim 1, wherein the cell signal orstimulus comprises TNF-a and IL-la.
 13. A composition for treating asubject having or suspected of having a disease or disorder, thecomposition comprising a modified cell comprising a modified endogenousgene, wherein an endogenous gene or fragment thereof comprises a signalpeptide and the signal peptide is deleted or knocked out using aCRISPR/Cas9 system to generate the modified endogenous gene, themodified cell having an altered response to a cell signal or stimulus.14. The composition of claim 13, wherein the altered response to thecell signal or stimulus comprises a decrease in responsiveness of themodified endogenous gene to the cell signal or stimuli compared to anunmodified endogenous gene.
 15. The composition of claim 13, wherein themodified cell is resistant to IL-1 induced inflammation.
 16. Thecomposition of claim 13, wherein the endogenous gene is IL lrl.
 17. Thecomposition of claim 13, wherein the cell signal or stimulus comprisesIL-1.
 18. The composition of claim 13, wherein the disease is a chronicdisease.
 19. The composition of claim 18, wherein the chronic disease isosteoarthritis.
 20. The composition of claim 13, wherein the disease iscancer. 21.-43. (canceled)